Unclassified

ENV/JM/MONO(2011)39

Organisation de Coopération et de Développement Économiques
Organisation for Economic Co-operation and Development

08-Sep-2011___________________________________________________________________________________________English - Or. English

ENVIRONMENT DIRECTORATEENV/JM/MONO(2011)39
Unclassified

JOINT MEETING OF THE CHEMICALS COMMITTEE AND
THE WORKING PARTY ON CHEMICALS, PESTICIDES AND BIOTECHNOLOGYVALIDATION REPORT FOR THE SKIN IRRITATION TEST METHODUSING LABCYTE EPI-MODEL24

English - Or. English

JT03306650
Document complet disponible sur OLIS dans son format d'origine
Complete document available on OLIS in its original format

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ENV/JM/MONO(2011)39

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ENV/JM/MONO(2011)39

OECD Environment, Health and Safety Publications
Series on Testing and Assessment
No. 159

VALIDATION REPORT FOR THE SKIN IRRITATION TEST METHOD USINGLABCYTE EPI-MODEL24

Environment Directorate
ORGANISATION FOR ECONOMIC CO-OPERATION AND DEVELOPMENT
Paris 2011
323

ENV/JM/MONO(2011)39
Also published in the Series on Testing and Assessment:
No. 1,
Guidance Document for the Development of OECD
Guidelines for Testing of Chemicals (1993; reformatted 1995, revised
2006)
No. 2,(1995)

Detailed Review Paper on Biodegradability Testing

No. 3,(1995)

Guidance Document for Aquatic Effects Assessment

No. 4,
Report of the OECD Workshop on Environmental
Hazard/Risk Assessment (1995)
No. 5,
Report of the SETAC/OECD Workshop on Avian Toxicity
Testing (1996)
No. 6,
Report of the Final Ring-test of the Daphnia magna
Reproduction Test (1997)
No. 7,
Guidance Document on Direct Phototransformation ofChemicals in Water (1997)
No. 8,
Report of the OECD Workshop on Sharing Information
about New Industrial Chemicals Assessment (1997)
No. 9,
Guidance Document for the Conduct of Studies ofOccupational Exposure to Pesticides during Agricultural Application(1997)
No. 10,
Report of the OECD Workshop on Statistical Analysis of
Aquatic Toxicity Data (1998)
No. 11,
Detailed Review Paper on Aquatic Testing Methods for
Pesticides and industrial Chemicals (1998)
No. 12,
Detailed Review Document on Classification Systems forGerm Cell Mutagenicity in OECD Member Countries (1998)
No. 13,
Detailed Review Document on Classification Systems for
Sensitising Substances in OECD Member Countries 1998)
No. 14,
Detailed Review Document on Classification Systems for
Eye Irritation/Corrosion in OECD Member Countries (1998)
No. 15,
Detailed Review Document on Classification Systems for
Reproductive Toxicity in OECD Member Countries (1998)
No. 16,
Detailed Review Document on Classification Systems for
Skin Irritation/Corrosion in OECD Member Countries (1998)
4

24

ENV/JM/MONO(2011)39
No. 17,
Environmental Exposure Assessment Strategies for
Existing Industrial Chemicals in OECD Member Countries (1999)
No. 18,
Report of the OECD Workshop on Improving the Use of
Monitoring Data in the Exposure Assessment of Industrial Chemicals(2000)
No. 19,
Guidance Document on the Recognition, Assessment andUse of Clinical Signs as Humane Endpoints for Experimental Animals
used in Safety Evaluation (1999)
No. 20,
Revised Draft Guidance Document for Neurotoxicity
Testing (2004)
No. 21,
Detailed Review Paper: Appraisal of Test Methods for
Sex Hormone Disrupting Chemicals (2000)
No. 22,
Guidance Document for the Performance of Out-door
Monolith Lysimeter Studies (2000)
No. 23,
Guidance Document on Aquatic Toxicity Testing ofDifficult Substances and Mixtures (2000)
No. 24,(2001)

Guidance Document on Acute Oral Toxicity TestingNo. 25,
Detailed Review Document on Hazard Classification
Systems for Specifics Target Organ Systemic Toxicity Repeated
Exposure in OECD Member Countries (2001)
No. 26,
Revised Analysis of Responses Received from Member
Countries to the Questionnaire on Regulatory Acute Toxicity Data
Needs (2001)
No 27,
Guidance Document on the Use of the Harmonised
System for the Classification of Chemicals which are Hazardous for the
Aquatic Environment (2001)
No 28,
Guidance Document for the Conduct of Skin Absorption
Studies (2004)
No 29,
Guidance Document on Transformation/Dissolution ofMetals and Metal Compounds in Aqueous Media (2001)
No 30,
Detailed Review Document on Hazard Classification
Systems for Mixtures (2001)
No 31,
Detailed Review Paper on Non-Genotoxic Carcinogens Detection: The
Performance of In-Vitro Cell Transformation Assays (2007)

525

ENV/JM/MONO(2011)39
No. 32,
Guidance Notes for Analysis and Evaluation of RepeatDose Toxicity Studies (2000)
No. 33,Harmonised Integrated Classification System for Human
Health and Environmental Hazards of Chemical Substances and
Mixtures (2001)
No. 34,
Guidance Document on the Development, Validation andRegulatory Acceptance of New and Updated Internationally Acceptable
Test Methods in Hazard Assessment (2005)
No. 35,
Guidance notes for analysis and evaluation of chronic
toxicity and carcinogenicity studies (2002)
No. 36,
Report of the OECD/UNEP Workshop on the use ofMultimedia Models for estimating overall Environmental Persistence
and long range Transport in the context of PBTS/POPS Assessment(2002)
No. 37,
Detailed Review Document on Classification Systems for
Substances Which Pose an Aspiration Hazard (2002)
No. 38,
Detailed Background Review of the Uterotrophic Assay
Summary of the Available Literature in Support of the Project of theOECD Task Force on Endocrine Disrupters Testing and Assessment(EDTA) to Standardise and Validate the Uterotrophic Assay (2003)
No. 39,
Guidance Document on Acute Inhalation Toxicity Testing(in preparation)
No. 40,Detailed Review Document on Classification in OECD
Member Countries of Substances and Mixtures Which Cause
Respiratory Tract Irritation and Corrosion (2003)
No. 41,
Detailed Review Document on Classification in OECD
Member Countries of Substances and Mixtures which in Contact with
Water Release Toxic Gases (2003)
No. 42,
Guidance Document on Reporting Summary Information
on Environmental, Occupational and Consumer Exposure (2003)
No. 43,
Guidance Document on Mammalian Reproductive
Toxicity Testing and Assessment (2008)
No. 44,
Description of Selected Key Generic Terms Used in
Chemical Hazard/Risk Assessment (2003)
No. 45,
Guidance Document on the Use of Multimedia Models for
Estimating Overall Environmental Persistence and Long-range
Transport (2004)
626

ENV/JM/MONO(2011)39
No. 46,
Detailed Review Paper on Amphibian Metamorphosis
Assay for the Detection of Thyroid Active Substances (2004)
No. 47,
Detailed Review Paper on Fish Screening Assays for the
Detection of Endocrine Active Substances (2004)
No. 48,New Chemical Assessment Comparisons and Implications
for Work Sharing (2004)
No. 49,
Report from the Expert Group on (Quantitative)
Structure-Activity Relationships [(Q)SARs] on the Principles for the
Validation of (Q)SARs (2004)
No. 50,(2005)

Report of the OECD/IPCS Workshop on Toxicogenomics

No. 51,
Approaches to Exposure Assessment in OECD Member
Countries: Report from the Policy Dialogue on Exposure Assessment inJune 2005 (2006)
No. 52,Comparison of emission estimation methods used in
Pollutant Release and Transfer Registers (PRTRs) and Emission
Scenario Documents (ESDs): Case study of pulp and paper and textile
sectors (2006)
No. 53,
Guidance Document on Simulated Freshwater Lentic
Field Tests (Outdoor Microcosms and Mesocosms) (2006)
No. 54,Current Approaches in the Statistical Analysis of
Ecotoxicity Data: A Guidance to Application (2006)
No. 55,
Detailed Review Paper on Aquatic Arthropods in Life Cycle
Toxicity Tests with an Emphasis on Developmental, Reproductive andEndocrine Disruptive Effects (2006)
No. 56,
Guidance Document on the Breakdown of Organic Matterin Litter Bags (2006)
No. 57,
Detailed Review Paper on Thyroid Hormone Disruption
Assays (2006)
No. 58,
Report on the Regulatory Uses and Applications in OECD
Member Countries of (Quantitative) Structure-Activity Relationship[(Q)SAR] Models in the Assessment of New and Existing Chemicals(2006)
No. 59,
Report of the Validation of the Updated Test Guideline
407: Repeat Dose 28-Day Oral Toxicity Study in Laboratory Rats
(2006)

7

27

ENV/JM/MONO(2011)39
No. 60,
Report of the Initial Work Towards the Validation of the
21-Day Fish Screening Assay for the Detection of Endocrine Active
Substances (Phase 1A) (2006)
No. 61,
Report of the Validation of the 21-Day Fish Screening
Assay for the Detection of Endocrine Active Substances (Phase 1B)(2006)
No. 62,
Final OECD Report of the Initial Work Towards the
Validation of the Rat Hershberger Assay: Phase-1, Androgenic
Response to Testosterone Propionate, and Anti-Androgenic Effects of
Flutamide (2006)
No. 63,

Guidance Document on the Definition of Residue (2006)

No. 64,
Guidance Document on Overview of Residue Chemistry
Studies (2006)
No. 65,
OECD Report of the Initial Work Towards the Validationof the Rodent Uterotrophic Assay - Phase 1 (2006)
No. 66,
OECD Report of the Validation of the Rodent
Uterotrophic Bioassay: Phase 2. Testing of Potent and Weak
Oestrogen Agonists by Multiple Laboratories (2006)
No. 67,
Additional data supporting the Test Guideline on the
Uterotrophic Bioassay in rodents (2007)
No. 68,
Summary Report of the Uterotrophic Bioassay Peer
Review Panel, including Agreement of the Working Group of the
National Coordinators of the Test Guidelines Programme on the follow
up of this report (2006)
No. 69,
Guidance Document on the Validation of (Quantitative)
Structure-Activity Relationship [(Q)SAR] Models (2007)
No. 70,
Report on the Preparation of GHS Implementation by theOECD Countries (2007)
No. 71,
Guidance Document on the Uterotrophic Bioassay Procedure to Test for Antioestrogenicity (2007)
No. 72,
Guidance Document on Pesticide Residue Analytical
Methods (2007)
No. 73,
Report of the Validation of the Rat Hershberger Assay:
Phase 3: Coded Testing of Androgen Agonists, Androgen Antagonists
and Negative Reference Chemicals by Multiple Laboratories. SurgicalCastrate Model Protocol (2007)

828

ENV/JM/MONO(2011)39
No. 74,
Detailed Review Paper for Avian Two-generation Toxicity
Testing (2007)
No. 75,
Guidance Document on the Honey Bee (Apis Mellifera L.)
Brood test Under Semi-field Conditions (2007)
No. 76,
Final Report of the Validation of the Amphibian
Metamorphosis Assay for the Detection of Thyroid Active Substances:
Phase 1 - Optimisation of the Test Protocol (2007)
No. 77,
Final Report of the Validation of the Amphibian
Metamorphosis Assay: Phase 2 - Multi-chemical Interlaboratory Study(2007)
No. 78,
Final Report of the Validation of the 21-day Fish
Screening Assay for the Detection of Endocrine Active Substances.
Phase 2: Testing Negative Substances (2007)
No. 79,Validation Report of the Full Life-cycle Test with the
Harpacticoid Copepods Nitocra Spinipes and Amphiascus Tenuiremis
and the Calanoid Copepod Acartia Tonsa - Phase 1 (2007)
No. 80,

Guidance on Grouping of Chemicals (2007)

No. 81,
Summary Report of the Validation Peer Review for theUpdated Test Guideline 407, and Agreement of the Working Group of
National Coordinators of the Test Guidelines Programme on thefollow-up of this report (2007)
No. 82,(2007)

Guidance Document on Amphibian Thyroid Histology

No. 83,
Summary Report of the Peer Review Panel on the Stably
Transfected Transcriptional Activation Assay for Detecting Estrogenic
Activity of Chemicals, and Agreement of the Working Group of the
National Coordinators of the Test Guidelines Programme on the
Follow-up of this Report (2007)
No. 84,
Report on the Workshop on the Application of the GHS
Classification Criteria to HPV Chemicals, 5-6 July Bern Switzerland(2007)
No. 85,
Report of the Validation Peer Review for the Hershberger
Bioassay, and Agreement of the Working Group of the National
Coordinators of the Test Guidelines Programme on the Follow-up of
this Report (2007)
No. 86,
Report of the OECD Validation of the Rodent
Hershberger Bioassay:
Phase 2: Testing of Androgen Agonists,
Androgen Antagonists and a 5 Į-Reductase Inhibitor in Dose Response
Studies by Multiple Laboratories (2008)
929

ENV/JM/MONO(2011)39
No. 87,
Report of the Ring Test and Statistical Analysis of
Performance of the Guidance on Transformation/Dissolution of Metals
and Metal Compounds in Aqueous Media (Transformation/ Dissolution
Protocol) (2008)
No. 88,Workshop on Integrated Approaches to Testing andAssessment (2008)
No. 89,Retrospective Performance Assessment of the Test
Guideline 426 on Developmental Neurotoxicity (2008)
No.90,
Background Review Document on the Rodent
Hershberger Bioassay (2008)
No. 91,
Report of the Validation of the Amphibian Metamorphosis
Assay (Phase 3) (2008)
No. 92,
Report of the Validation Peer Review for the Amphibian
Metamorphosis Assay and Agreement of the Working Group of the
National Coordinators of the Test Guidelines Programme on the
Follow-Up of this Report (2008)
No. 93,
Report of the Validation of an Enhancement of OECD TG
211: Daphnia Magna Reproduction Test (2008)
No. 94,
Report of the Validation Peer Review for the 21-Day Fish
Endocrine Screening Assay and Agreement of the Working Group of
the National Coordinators of the Test Guidelines Programme on the
Follow-up of this Report (2008)
No. 95,

Detailed Review Paper on Fish Life-Cycle Tests (2008)

No.96,
Guidance Document on Magnitude of Pesticide Residues
in Processed Commodities (2008)
No.97,
Detailed Review Paper on the use of Metabolising
Systems for In Vitro Testing of Endocrine Disruptors (2008)
No. 98,
Considerations Regarding Applicability of the Guidanceon Transformation/Dissolution of Metals Compounds in Aqueous
Media (Transformation/Dissolution Protocol) (2008)
No. 99,Comparison between OECD Test Guidelines and ISO
Standards in the Areas of Ecotoxicology and Health Effects (2008)
No. 100,(2009)

Report of the Second Survey on Available Omics Tools

No. 101, Report of the Workshop on Structural Alerts for the OECD
(Q)SAR Application Toolbox, 15-16 May 2008, Utrecht, theNetherlands (2009)1030

ENV/JM/MONO(2011)39
No. 102, Guidance Document for using the OECD (Q)SAR
Application Toolbox to Develop Chemical Categories According to theOECD Guidance on Grouping of Chemicals (2009)
No. 103, Detailed Review Paper on Transgenic Rodent Mutation
Assays (2009)
No. 104, Performance Assessment: Comparsion of 403 and CxTProtocols via Simulation and for Selected Real Data Sets (2009)
No. 105, Report on Biostatistical Performance Assessment of thedraft TG 436 Acute Toxic Class Testing Method for Acute Inhalation
Toxicity (2009)
No. 106, Guidance Document for Histologic Evaluation of Endocrine
and Reproductive Test in Rodents (2009)
No. 107, Preservative treated wood to the environment for wood heldin storage after treatment and for wooden commodities that are notcover and are not in contact with ground. (2009)
No. 108, Report of the validation of the Hershberger Bioassay(weanling model) (2009)
No. 109, Literature review on the 21-Day Fish Assay and the Fish
Short-Term Reproduction Assay (2009)
No. 110, Report of the validation peer review for the weanling
Hershberger Bioassay and agreement of the working of national
coordinators of the test guidelines programme on the follow-up of thisreport (2009)
No. 111, Report of the Expert Consultation to Evaluate an Estrogen
Receptor Binding Affinity Model for Hazard Identification (2009)
No. 112, The 2007 OECD List of High Production Volume Chemicals(2009)
No. 113, Report of The Focus Session on Current and Forthcoming
Approaches for Chemical Safety and Animal Welfare (2010)
No. 114, Performance Assessment of Different Cytotoxic andCytostatic Measures for the In Vitro Micronucleus Test (MNVIT):
Summary of results in the collaborative trial (2010)
No. 115, Guidance Document on the Weanling Hershberger Bioassay
in Rats: A Short-term Screening Assay for (Anti) Androgenic Properties(2009)

1131

ENV/JM/MONO(2011)39
No. 116, Guidance Document on the Design and Conduct of Chronic
Toxicity and Carcinogenicity Studies, Supporting TG 451, 452 and 453(2010)
No. 117, Guidance Document 117 on the Current Implementation ofInternal Triggers in Test Guideline 443 for an Extended One
Generation Reproductive Toxicity Study, in the United States andCanada (2011)
No. 118, Workshop Report on OECD Countries Activities Regarding
Testing, Assessment and Management of Endocrine Disrupters Part I
and Part II (2010)
No. 119, Classification and Labelling of chemicals according to the
UN Globally Harmonized System: Outcome of the Analysis ofClassification of Selected Chemicals listed in Annex III of theRotterdam Convention (2010)
No. 120, Part 1: Report of the Expert Consultation on Scientific andRegulatory Evaluation of Organic Chemistry Mechanism-based
Structural Alerts for the Identification of DNA Binding Chemicals
No. 120, Part 2: Report of the Expert Consultation on Scientific andRegulatory Evaluation of Organic Chemistry Mechanism-based
Structural Alerts for the Identification of DNA Binding Chemicals
No. 121, Detailed review paper (DRP) on Molluscs life-cycle Toxicity
Testing (2010)
No. 122, Guidance Document on the determination of the Toxicity ofa Test Chemical to the Dung Beetle Aphodius Constans (2010)
No. 123, Guidance Document on the Diagnosis of Endocrine-related
Histopathology in Fish Gonads (2010)
No. 124,(2010)

Guidance for the Derivation of an Acute Reference DoseNo. 125, Guidance Document on Histopathology for Inhalation
Toxicity Studies, Supporting TG 412 (Subacute Inhalation Toxicity: 28Day) and TG 413 (Subchronic Inhalation Toxicity: 90-Day) (2010)
No. 126, Short Guidance on the Threshold approach for Acute Fish
Toxicity (2010)
No. 127, Peer review report of the validation of the 21-day
androgenised female stickleback screening assay (2010)
No. 128, Validation Report of the 21-day Androgenised Female
Stickleback Screening Assay (2010)12

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ENV/JM/MONO(2011)39
No. 129, Guidance Document on using Cytotoxicity Tests toEstimate Starting Doses for Acute Oral Systemic Toxicity Tests(2010)
No. 131, Report of the Test Method Validation of Avian Acute Oral
Toxicity Test (OECD test guideline 223) (2010)
No. 132, Report of the Multi-Laboratory Validation of the H295R
Steroidogenesis Assay to Identify Modulators (2010)
No.133, Peer Review Report for the H295R Cell-Based Assay for
Steroidogenesis (2010)
No.134, Report of the Validation of a Soil Bioaccumulation Test with
Terrestrial Oligochaetes by an International ring test (2010)
No.135, Detailed Review Paper on Environmental Endocrine
Disruptor Screening: The use of Estrogen and Androgen Receptor
Binding and Transactivation Assays in Fish (2010)
No. 136, Validation Report of t he Chironomid Full Life-Cycle
Toxicity Test (2010)
No. 137, Explanatory Background Document to the OECD Test
Guideline On In Vitro Skin Irritation Testing (2010)
No. 138, Report of the Workshop on Using Mechanistic Informationin Forming Chemical Categories (2011)
No. 139, Report of the Expert Consultation on Scientific andRegulatory Evaluation of Organic Chemistry Mechanism Based
Structural Alerts for the Identification of Protein-binding Chemicals(2011)
No. 141, Report of the Phase 1 of the Validation of the Fish Sexual
Development Test for the Detection of Endocrine Active Substances(2011)
No. 142, Report of the Phase 2 of the Validation of the Fish Sexual
Development Test for the Detection of Endocrine Active Substances(2011)
No. 143, Peer Review Report for the Validation of the Fish Sexual
Development Test and Agreement of the Working Group of National
Co-ordinators of the Test Guideline Programme on the Follow-up of
the Peer Review (2011)
No. 144,

Validation Report for the Acute Chironomid Assay (2011)13

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ENV/JM/MONO(2011)39
No. 145, Transgenic Rodent Somatic and Germ Cell Gene Mutation Assay:
Retrospective Performance Assessment (2011)
No. 148, Guidance Document on the Androngenised Female
Stickleback Screen (2011)
No. 152, Case Study: Assessment of an Extended Chemical Category,
the Short-chain Methacrylates, Targeted on Bioaccumulation (2011)
No. 153, Guidance Document for the Derivation of an Acute
Reference Concentration (Arfc) (2011)
No. 156,

Guidance Notes on Dermal Absorption (2011)

No. 157, Validation Report Phase 1 for the Zebrafish Embryo Toxicity
Test (2011)
No. 158, Report of Progress on the Interlaboratory Validation of theOECD Harpacticoid Copepod Development and Reproduction Test
(2011)
No. 159, Validation Report for the Skin Irritation Test Method using
Labcyte Epi-Model24(2011)© OECD 2011
Applications for permission to reproduce or translate all or part of thismaterial should be made to: Head of Publications Service,
RIGHTS@oecd.org. OECD, 2 rue André-Pascal, 75775 Paris Cedex
16, France

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About the OECDThe Organisation for Economic Co-operation and Development (OECD) is an intergovernmentalorganisation in which representatives of 34 industrialised countries in North and South America, Europe
and the Asia and Pacific region, as well as the European Commission, meet to co-ordinate and harmonise
policies, discuss issues of mutual concern, and work together to respond to international problems. Most of
the OECD’s work is carried out by more than 200 specialised committees and working groups composedof member country delegates. Observers from several countries with special status at the OECD, and frominterested international organisations, attend many of the OECD’s workshops and other meetings.
Committees and working groups are served by the OECD Secretariat, located in Paris, France, which isorganised into directorates and divisions.
The Environment, Health and Safety Division publishes free-of-charge documents in ten different series:
Testing and Assessment; Good Laboratory Practice and Compliance Monitoring; Pesticides andBiocides; Risk Management; Harmonisation of Regulatory Oversight in Biotechnology; Safety ofNovel Foods and Feeds; Chemical Accidents; Pollutant Release and Transfer Registers; Emission
Scenario Documents; and Safety of Manufactured Nanomaterials. More information about theEnvironment, Health and Safety Programme and EHS publications is available on the OECD’s World
Wide Web site (www.oecd.org/ehs/).

This publication was developed in the IOMC context. The contents do not necessarily reflect the views orstated policies of individual IOMC Participating Organisations.
The Inter-Organisation Programme for the Sound Management of Chemicals (IOMC) wasestablished in 1995 following recommendations made by the 1992 UN Conference on Environmentand Development to strengthen co-operation and increase international co-ordination in the field ofchemical safety. The Participating Organisations are FAO, ILO, UNEP, UNIDO, UNITAR, WHO,
World Bank and OECD. UNDP is an observer. The purpose of the IOMC is to promote coordination of the policies and activities pursued by the Participating Organisations, jointly or
separately, to achieve the sound management of chemicals in relation to human health and theenvironment.

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ENV/JM/MONO(2011)39

This publication is available electronically, at no charge.
For this and many other Environment,
Health and Safety publications, consult the OECD’s
World Wide Web site (www.oecd.org/ehs/)
or contact:
OECD Environment Directorate,
Environment, Health and Safety Division
2 rue André-Pascal
75775 Paris Cedex 16
FranceFax: (33-1) 44 30 61 80 E-mail: ehscont@oecd.org
E-mail: ehscont@oecd.org

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ENV/JM/MONO(2011)39

FOREWORD
This document presents the validation report for the “Skin Irritation Test Using the LabCyte EPIMODEL24”. The project for developing a Test Guideline for an in vitro epidermal model to assess skinirritation using the LabCyte EPI-MODEL24, led by Japan, was included in the work plan of the Test
Guidelines Programme in 2009. The Working of National Coordinators of the Test Guidelines Programme
endorsed this validation at its meeting held on 12-14 April 2011. The Joint Meeting of the Chemicals
Committee and Working Party on Chemicals, Pesticides and Biotechnology (Joint Meeting) agreed to its
declassification on 5 August 2011.
A validation peer review report, accompanied by a report on additional validation work, is also expected tobe published in the Series on Testing and Assessment.
This document is published under the responsibility of the Joint Meeting.

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ENV/JM/MONO(2011)39

MEMBERS OF LABCYTE VALIDATION MANAGEMENT TEAM
Mr. Hajime Kojima, JaCVAM: Japanese Centre for the Validation of Alternative Methods
Mr. Takashi Omori; Kyoto Univ.
Mr. Kenji Idehara; Daicel Chemical Co.
Mr. Isao Yoshimura; Tokyo University of Science
Mr.Etsuyoshi Miyaoka; Tokyo University of Science
Mr. Masakazu Kato; Japan Tissue Engineering Co. Ltd.
Mr. Toshiro Yokouchi; Japan Tissue Engineering Co. Ltd.

Participant laboratoriesx

Aiken Co., Ltd. (Ms Yoko Ando and Ms Yui Asako)x

KOBAYASHI Pharmaceutical Co., Ltd. (Mr. Yoshihiro Yamaguchi and Ms Maki Nakamura)x

The Institute of Environmental Toxicology (Mr Tadashi Kosaka and Mr Koichi hayashi)x

Fancl Corp. (Ms. Tamie Suzuki and Ms Runa Izumi)x

FUJIFILM Corporation (Ms. Atsuko Yuasa, and Mr Shinichi Akimoto)x

Maruishi Pharmaceutical Co., Ltd. (Mr Yukihiko Watanabe and Mr Osamu Mitani)x

Drug Safety Testing Center Co., Ltd. (Mr Shinsuke Shinoda and Ms Saori Hagiwara)

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ENV/JM/MONO(2011)39
TABLE OF CONTENTS
1. Goal Statement21

2. Objective

21

3. Test Methods

21

3-1. Reconstructed human cultured dermal model

21

3-2. Model supplier21

4. Validation Management Structure224-1. Validation Management Team22

4-2. Chemical selection, acquisition, coding and distribution

22

4-3. Independent biostatisticians

22

4-4. Participating laboratories

23

4-5. Sponsorship24

5. Study Design24

6. Test Chemicals

246-1. Chemical selection

24

6-2. Deficit chemical

26
26

6-3. Chemical coding and distribution

26

7. Protocol
7-1. Protocol of the skin irritation test with LabCyte EPI-MODEL24
7-2. Prediction model of skin irritation

26
27

7-3. Difference between LabCyte EPI-MODEL 24 protocol and EPISKIN 27
protocol7-4. Data collection, handling, and analysis

28

7-5. Quality assurance, GLP

28

8. Results

29

8-1. 1st Phase

29

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ENV/JM/MONO(2011)39
8-1-1. Negative Control

29

8-1-2. Positive control and test chemicals
8-2. 2nd phase & 3rd phase

29
30
30

8-2-1. Comments on the Datasheets
8-2-2. Negative control

32

8-2-3. Positive control

33
34

8-2-4. Skin irritation test by cell viability
8-2-5. IL-1Į

43

8-2-6. Classification of three independent viabilities at each laboratory45

9. Discussion

47
47

9-1. Reliability
9-2. Predictivity
10. Conclusions

48
48

11. Acknowledgements

48

12. References

49

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ENV/JM/MONO(2011)39
1. Goal statementx

The aim of this study was to validate in vitro skin irritation tests in a formal inter-laboratory study,
the ultimate goal of the test strategy will be to replace the regulatory Draize skin irritation test
according OECD TG 404 (OECD, 2002).

x

The primary goal of this validation study was an evaluation of the ability of the in vitro tests toreliably discriminate skin irritant (I) from non-irritant (NI) chemicals, as defined according to theOECD and United Nations proposal for Globally Harmonised System (GHS) for the classificationand labelling of skin irritation (category 1/category 2; no category; Anon., 2003) .

2. Objective
1.
The in vitro test system, employing reconstructed human epidermis model (RhE: LabCyte EPIMODEL24), has progressed through protocol optimisation as in vitro skin irritation test. The multilaboratory assessment of this system performed based on the a few ECVAM performance standards (ESAC
statement, 2007, 2008, 2009). This report shows the results of 3rd phase validation study in accordance withthe revised reference chemicals described by the new ESAC statement 2009.
2.
The present objective was to conduct a validation study to assess the reliability (reproducibility
within and between laboratories) and relevance (predictive capacity) of this test system with a challenging
set of coded 25 test chemicals for which high quality in vivo data were available. The validation study was
undertaken in accordance with the principles and criteria documented in the OECD Guidance Document on
the Validation and International Acceptance of New or Updated Test Methods for Hazard Assessment (No.
34, OECD, 2005) and according to the Modular Approach to validation (Hartung et al. 2004).
3. Test Method
3-1. Reconstructed human cultured epidermal model
3.
LabCyte EPI-MODEL24 is a new, commercially available RhE model produced by Japan Tissue
Engineering Co. Ltd. It consists of normal human epidermal keratinocytes whose biological origin isneonate foreskin. In order to expand human keratinocytes while maintaining their phenotype, they were
cultured with 3T3-J2 cells as a feeder layer (Rheinwald and Green, 1975; Green, 1978). Reconstruction ofhuman cultured epidermis is achieved by cultivating and proliferating keratinocytes on an inert filter
substrate (surface 0.3 cm2) at the air-liquid interface for 13 days with an optimized medium containing 5%
fetal bovine serum. It constructs a multilayer structure consisting of a fully differentiated epithelium withfeatures of the normal human epidermis, including a stratum corneum. LabCyte EPI-MODEL24 is embedded
in an agarose gel containing nutrient solution and shipped in 24-well plates at around 18°C (Kato, 2009).
3-2. MODEL SUPPLIER
4.
According to OECD GLP Consensus Document No.5 “Compliance of Laboratory Suppliers with
GLP Principles” the responsibility for the quality and fitness for use of equipment and materials rests
entirely with the management of the test facility (OECD, 1999).
5.
The acceptability of equipment and materials in laboratories complying with GLP-like principles
should therefore be guaranteed to any regulatory authority to which studies were submitted. In somecountries where GLP has been implemented, suppliers belong to national regulatory or voluntaryaccreditation schemes (for laboratory animals) which can provide users with additional documentary
evidence that they are using a test system of a defined quality.
6.
The audits focused on the procedures established to guarantee a defined quality of the tissue
models.

21
41

ENV/JM/MONO(2011)39
4. Validation Management structure
7.
This validation study was performed by the Japanese Society for the Alternative to Animal
Experiments (JSAAE).
The management structure of the study is shown in Figure 1.
4-1. Validation Management Group8.
The Validation Management Team (VMT), which plays a central role overseeing the conduct ofthe validation study, includes:
1) Goal statement
2) Project plan including objective3) Study protocol / amendments
4) Outcome of QC audits5) Test chemicals
6) Data management procedures
7) Timeline/ study progression8) Study interpretation and conclusions
9) Reports and publication
9.
The final decision on which laboratories participate in the validation study is the responsibility of
the VMT.
Members:
A chair (Hajime Kojima, JaCVAM: Japanese Centre for the Validation of Alternative Methods)
The sponsor representative: representatives of JSAAE (Takashi Omori; Kyoto Univ., Kenji Idehara;
Daicel Chemical Co. and Isao Yoshimura; Tokyo University of Science)
The sponsor representative, LabCyte EPI-MODEL24suppliers and lead lab (Masakazu Kato : Japan
Tissue Engineering Co., Ltd, J-TEC)
4-2. Chemical selection, acquisition, coding and distribution1) Definition of selection criteria
2) Chemical selection3) Liaise with suppliers
4) Final check of chemicals provided
5) Acquisition
6) Coding7) Distribution
MemberHajime Kojima, JaCVAM
4-3 . Independent biostatisticians
1) Approve spreadsheets
2) Collect data
3) Analyse data
Members:
Takashi Omori: Kyoto Univ., Etsuyoshi MIyaoka and Kenya Ishiyama: Tokyo University of Science2242ENV/JM/MONO(2011)39

Validation
TeamTeam
ValidationManagement
Management

Chair: JaCVAM
JaCVAM
Chair:
Biostatistics

Study Sponser
JSAAE
JSAAE

Kyoto Univ.
Kyoto Univ.
Tokyo Universityof Science
Tokyo
Universityof Science

Chemical coding
and distribution
and distribution
JaCVAM
JaCVAM
JaCVAM

Lead
laboratorylaboratory&&
Training
J-TEC
Training
J-TEC

Aiken
Aiken
Co.,
Co.
Ltd.

KOBAYASHI
PharmaceuPharmaceut
tical
Co.,
ical
Ltd.
Co.

The Institute ofEnvironmental
Environmental
Toxicology
Toxicology

Fancl㩷Corp.
Corp.

FUJIFILM
Corporation
Corporation

MaruishiPharmaceutiPharmaceuticcal Co.,al Ltd.
Co.

Drug
Drug
SafetySafetyTesting
Testing
CenterCo.,
CenterCo., Ltd.
Ltd.

Figure 1. Management structure of the JSAAE skin irritation validation study
4-4. Participating laboratories
The laboratories participating in the study are to be defined as shown in Fig. 1.
The following 6 laboratories participated in the validation study for the evaluation of the LabCyte
EPI-MODEL24 assays:
x Laboratory 1 – Aiken Co., Ltd. (Yoko Ando and Yui Asako)x

Laboratory 2 – KOBAYASHI Pharmaceutical Co., Ltd. (Yoshihiro Yamaguchi and Maki
Nakamura)x

Laboratory 3 – The Institute of Environmental Toxicology (Tadashi Kosaka and Koichi
hayashi)x

Laboratory 4 – FanclG Corp. (Tamie Suzuki and Runa Izumi)xLaboratory 5 – FUJIFILM Corporation (Atsuko Yuasa, and Shinichi Akimoto)x

This laboratory was not participated at the 3rd Phase study.
Laboratory 6 – Maruishi Pharmaceutical Co., Ltd. (Yukihiko Watanabe and Osamu Mitani)x

Laboratory 7 – Drug Safety Testing Center Co., Ltd. (Shinsuke Shinoda and Saori Hagiwara)

A lead laboratory is also identified as J-TEC (Mr. Masakazu Kato and Mr Toshihiro Yokouchi). This
laboratory was not participated in the validation study.
Each laboratory also was responsible for complying with GLP-like principles and specifying QA aspects.

2343

ENV/JM/MONO(2011)39

4-5. Sponsorship
The study was managed and finance by JSAAE and J-TEC .
1) JSAAE finance:- the management of the study (VMT meetings)- the independent statistical support (biostatistician)- the responsible for the chemicals purchase, coding and distribution to the laboratories- the independent QC audit of the data
- the publication of the study2) J-TECG finance:- the lead laboratories for the test method- training for the participating laboratories- the independent QC audit on the LabCyte EPI-MODEL24- the financial assistance for the participated laboratories
5. Study design10.
Before this validation study, the training course using LabCyte EPI-MODEL24 was performed by
J-TEC on April, 2008. All technicians from each laboratory participated at this training course.
11.
Three phases of validation studies were performed. In the 1st phase, we confirmed thetransferability of the test protocol and assessed its reproducibility, by testing three coded chemicals (ethanol,
glycerol and napthalen acetic acid) and a positive control (5% sodium lauryl sulfate solution) in sevenlaboratories between June and July of 2008.
12.
In the 2nd phase study, we confirmed the intra- and inter-laboratory reproducibility robustness, andthe correlation of test using 19 new chemicals tested in reference to the original EPISKIN performancestandards (ECVAM, 2007) . These tests were conducted by 7 laboratories between September 2008 andJanuary of 2009.
13.
In the 3rd phase study, we confirmed the intra- and inter-laboratory reproducibility robustness, andthe correlation of test using 6 chemicals tested in reference to the new EPISKIN performance standards(ESAC statement, 2009). This study was conducted by 6 laboratories, which attend the 1st and 2nd phasevalidation study between April to May, 2009.
6. Test Chemical
6-1. Chemicals Selection and list
14.
In 1st phase study, JaCVAM selected three coded chemicals (ethanol, glycerol and napthalen aceticacid) to test.
15.
According to the original ESAC Performance Standard (ESAC statement,2007) in 2nd Phase, theVMT selected 19 new chemicals to test in Table 1. One chemical, tri-isobuthyl phosphate (No. 13) on thechemical list reference for the original ECVAM Performance Standard cannot be purchased on the Japanesemarket. The VMT is responsible for the final approval of the chemicals proposed by JaCVAM. To avoid any
potential bias in the final selection, the laboratory representatives on the VMT were not party to these
discussions, nor were they informed of the final list of test chemicals for either phase of the validation study.
24
44

ENV/JM/MONO(2011)39
16.
According to the new ECVAM performance standard (ESAC statement, 2009) in 3rd phase, theVMT selected 6 new chemicals tested in Table 2. The final approval of the chemicals proposed by JaCVAM
is the responsibility of the VMT. To avoid any potential for bias in the final selection, the laboratory
representatives on the VMT did not be party to these discussions, nor were they made aware of the chemicals
finally approved for testing in either phase of the validation study.
Table 1. Reference test chemicals and codes㪥㫆㪅㪚㪿㪼㫄㫀㪺㪸㫃

㪠㫅㩷㫍㫀㫍㫆
㪚㪘㪪㪞㪟㪪㩷㫃㪸㪹㪼㫃 㫊㪺㫆㫉㪼㫅㫌㫄㪹㪼㫉
㩿㪧㪠㪠㪀㪣㪸㪹㫆㫉㪸㫋㫆㫉㫐
㪸

㪹

㪺

㪻

㪼

㪽

㪾

㪇㪈

㪈㪄㪹㫉㫆㫄㫆㪄㪋㪄㪺㪿㫃㫆㫉㫆㪹㫌㫋㪸㫅㪼 㪍㪐㪋㪇㪄㪎㪏㪄㪐㫅㫆㪇㪘㪄㪇㪈 㪙㪄㪇㪐㪐 㪚㪄㪇㪎㪎 㪛㪄㪈㪈㪌 㪜㪄㪈㪊㪊 㪝㪄㪇㪊㪈 㪞㪄㪇㪋㪐㪇㪉㪻㫀㪼㫋㪿㫐㫃㩷㫇㪿㫋㪿㪸㫃㪸㫋㪼㪏㪋㪄㪍㪍㪄㪉

㫅㫆㪇㪘㪄㪇㪉 㪙㪄㪈㪇㪇 㪚㪄㪇㪎㪏 㪛㪄㪈㪈㪍 㪜㪄㪈㪊㪋 㪝㪄㪇㪊㪉 㪞㪄㪇㪌㪇㪇㪊㪻㫀㪄㫇㫉㫆㫇㫐㫃㪼㫅㪼㩷㪾㫃㫐㪺㫆㫃 㪉㪌㪉㪍㪌㪄㪎㪈㪄㪏㫅㫆㪇㪘㪄㪇㪊 㪙㪄㪇㪏㪈 㪚㪄㪇㪎㪐 㪛㪄㪈㪈㪎 㪜㪄㪈㪊㪌 㪝㪄㪇㪊㪊 㪞㪄㪇㪌㪈㪇㪋㫅㪸㫇㪿㫋㪸㫃㪼㫅㩷㪸㪺㪼㫋㫀㪺㩷㪸㪺㫀㪻㪏㪍㪄㪏㪎㪄㪊

㫅㫆㪇㪘㪄㪇㪋 㪙㪄㪇㪏㪉 㪚㪄㪇㪏㪇 㪛㪄㪈㪈㪏 㪜㪄㪈㪊㪍 㪝㪄㪇㪊㪋 㪞㪄㪇㪌㪉㪇㪌㪸㫃㫃㫐㫃㩷㫇㪿㪼㫅㫆㫏㫐㪄㪸㪺㪼㫋㪸㫋㪼 㪎㪋㪐㪊㪄㪎㪋㪄㪌

㫅㫆

㪇㪅㪊

㪘㪄㪇㪌 㪙㪄㪇㪏㪊 㪚㪄㪇㪍㪈 㪛㪄㪈㪈㪐 㪜㪄㪈㪊㪎 㪝㪄㪇㪊㪌 㪞㪄㪇㪌㪊㪇㪍㫀㫊㫆㫇㫉㫆㫇㪸㫅㫆㫃㪍㪎㪄㪍㪊㪄㪇

㫅㫆㪇㪅㪊㪘㪄㪇㪍 㪙㪄㪇㪏㪋 㪚㪄㪇㪍㪉 㪛㪄㪈㪉㪇 㪜㪄㪈㪊㪏 㪝㪄㪇㪊㪍 㪞㪄㪇㪌㪋㪇㪎㪋㪄㫄㪼㫋㪿㫐㫃㪄㫋㪿㫀㫆㪄㪹㪼㫅㫑㪸㫃㪻㪼㪿㫐㪻㪼㪊㪋㪋㪍㪄㪏㪐㪄㪎㫅㫆㪈㪘㪄㪇㪎 㪙㪄㪇㪏㪌 㪚㪄㪇㪍㪊 㪛㪄㪈㪇㪈 㪜㪄㪈㪊㪐 㪝㪄㪇㪊㪎 㪞㪄㪇㪌㪌㪇㪏㫄㪼㫋㪿㫐㫃㩷㫊㫋㪼㪸㫉㪸㫋㪼㪈㪈㪉㪄㪍㪈㪄㪏

㫅㫆㪈㪘㪄㪇㪏 㪙㪄㪇㪏㪍 㪚㪄㪇㪍㪋 㪛㪄㪈㪇㪉 㪜㪄㪈㪋㪇 㪝㪄㪇㪊㪏 㪞㪄㪇㪌㪍㪇㪐㪸㫃㫃㫐㫃㩷㪿㪼㫇㫋㪸㫅㫆㪸㫋㪼㪈㪋㪉㪄㪈㪐㪄㪏

㫅㫆㪈㪅㪎㪘㪄㪇㪐 㪙㪄㪇㪏㪎 㪚㪄㪇㪍㪌 㪛㪄㪈㪇㪊 㪜㪄㪈㪉㪈 㪝㪄㪇㪊㪐 㪞㪄㪇㪌㪎㪈㪇㪿㪼㫇㫋㫐㫃㩷㪹㫌㫋㫐㫉㪸㫋㪼 㪌㪏㪎㪇㪄㪐㪊㪄㪐

㫅㫆

㪈㪅㪎

㪘㪄㪈㪇 㪙㪄㪇㪏㪏 㪚㪄㪇㪍㪍 㪛㪄㪈㪇㪋 㪜㪄㪈㪉㪉 㪝㪄㪇㪋㪇 㪞㪄㪇㪌㪏㪈㪈㪿㪼㫏㫐㫃㩷㫊㪸㫃㫀㪺㫐㫃㪸㫋㪼 㪍㪉㪌㪐㪄㪎㪍㪄㪊㫅㫆㪉㪘㪄㪈㪈 㪙㪄㪇㪏㪐 㪚㪄㪇㪍㪎 㪛㪄㪈㪇㪌 㪜㪄㪈㪉㪊 㪝㪄㪇㪉㪈 㪞㪄㪇㪌㪐㪈㪉㫋㪼㫉㫇㫀㫅㫐㫃㩷㪸㪺㪼㫋㪸㫋㪼㫅㫆㪉㪘㪄㪈㪉 㪙㪄㪇㪐㪇 㪚㪄㪇㪍㪏 㪛㪄㪈㪇㪍 㪜㪄㪈㪉㪋 㪝㪄㪇㪉㪉 㪞㪄㪇㪍㪇㪈㪊㪌㩿㪮㪆㪭㩷㩼㪀㩷㪪㪣㪪㪈㪋㪈㪄㪻㪼㪺㪸㫅㫆㫃㪏㪇㪄㪉㪍㪄㪉

㪘㪄㪈㪊 㪙㪄㪇㪐㪈 㪚㪄㪇㪍㪐 㪛㪄㪈㪇㪎 㪜㪄㪈㪉㪌 㪝㪄㪇㪉㪊 㪞㪄㪇㪋㪈㪈㪈㪉㪄㪊㪇㪄㪈 㪚㪸㫋㪼㪾㫆㫉㫐㩷㪉㪉㪅㪊㪘㪄㪈㪋 㪙㪄㪇㪐㪉 㪚㪄㪇㪎㪇 㪛㪄㪈㪇㪏 㪜㪄㪈㪉㪍 㪝㪄㪇㪉㪋 㪞㪄㪇㪋㪉㪈㪌㪺㫐㪺㫃㪸㫄㪼㫅㩷㪸㫃㪻㪼㪿㫐㪻㪼 㪈㪇㪊㪄㪐㪌㪄㪎 㪚㪸㫋㪼㪾㫆㫉㫐㩷㪉㪉㪅㪊㪘㪄㪈㪌 㪙㪄㪇㪐㪊 㪚㪄㪇㪎㪈 㪛㪄㪈㪇㪐 㪜㪄㪈㪉㪎 㪝㪄㪇㪉㪌 㪞㪄㪇㪋㪊㪈㪍㪈㪄㪹㫉㫆㫄㫆㪿㪼㫏㪸㫅㪼㪈㪈㪈㪄㪉㪌㪄㪈 㪚㪸㫋㪼㪾㫆㫉㫐㩷㪉㪉㪅㪎㪘㪄㪈㪍 㪙㪄㪇㪐㪋 㪚㪄㪇㪎㪉 㪛㪄㪈㪈㪇 㪜㪄㪈㪉㪏 㪝㪄㪇㪉㪍 㪞㪄㪇㪋㪋㪈㪎㱍㪄㫋㪼㫉㫇㫀㫅㪼㫆㫃㪐㪏㪄㪌㪌㪄㪌 㪚㪸㫋㪼㪾㫆㫉㫐㩷㪉㪉㪅㪎㪘㪄㪈㪎 㪙㪄㪇㪐㪌 㪚㪄㪇㪎㪊 㪛㪄㪈㪈㪈 㪜㪄㪈㪉㪐 㪝㪄㪇㪉㪎 㪞㪄㪇㪋㪌㪈㪏㪻㫀㪄㫅㪄㫇㫉㫆㫇㫐㫃㩷㪻㫀㫊㫌㫃㫇㪿㫀㪻㪼 㪍㪉㪐㪄㪈㪐㪄㪍 㪚㪸㫋㪼㪾㫆㫉㫐㩷㪉㪊㪘㪄㪈㪏 㪙㪄㪇㪐㪍 㪚㪄㪇㪎㪋 㪛㪄㪈㪈㪉 㪜㪄㪈㪊㪇 㪝㪄㪇㪉㪏 㪞㪄㪇㪋㪍㪈㪐㪹㫌㫋㫐㫃㩷㫄㪼㫋㪿㪸㪺㫉㫐㫃㪸㫋㪼㪊㪘㪄㪈㪐 㪙㪄㪇㪐㪎 㪚㪄㪇㪎㪌 㪛㪄㪈㪈㪊 㪜㪄㪈㪊㪈 㪝㪄㪇㪉㪐 㪞㪄㪇㪋㪎㪈㪈㪈㪄㪎㪈㪄㪎 㪚㪸㫋㪼㪾㫆㫉㫐㩷㪉㪋㪉㪇㪿㪼㫇㫋㪸㫅㪸㫃
1) CAS No.: Chemical abstracts service registry number.
2) PII: Primary irritation index.

㪘㪄㪉㪇 㪙㪄㪇㪐㪏 㪚㪄㪇㪎㪍 㪛㪄㪈㪈㪋 㪜㪄㪈㪊㪉 㪝㪄㪇㪊㪇 㪞㪄㪇㪋㪏㪐㪎㪄㪏㪏㪄㪈 㪚㪸㫋㪼㪾㫆㫉㫐㩷㪉

25
45

ENV/JM/MONO(2011)39
Table 2.G Test chemicals and code.
No.

ChemicalCASnumberA

Cinnamaldehyde

104-55-2B

2-Chroromethyl-3,5dimethyl-4-methoxypyridineHCl322-76821

C

Potassium hydroxide (5%aq)

168-21815

D
E
F

Benzenethiol, 5-(1,1dimethyethyl)-2-methyl
1-Methyl-3-phenyl-1piperazine
1,1,1-Torichloroethane

7340-90-1
5271-27-2
200-02463

GHS
label
no
Category
2
Category
2
Category
2
Category
2
Category
2

In
vivo
Score
2
2.7
3
3.3
3.3
4

Laboratory
a
A151

b
B176

c
C196

d
D216

f
F236

g
G256

A154

B173

C192

D211

F233

G253

A156
A153
A152
A155

B175
B172
B171
B174

C194
C191
C195
C193

D213
D214
D215
D212

F232
F234
F235
F231

G251
G254
G255
G252

1)CAS No.: Chemical abstracts service registry number.

6-2. Deficit chemical
17.
In Table1, tri-isobuthyl phosphate (No. 13) could not be used in the examination because it was notavailable in Japan. Therefore, a 5% SLS solution was used instead of tri-isobuthyl phosphate. The dataobtained with the 5% SLS solution were not used for calculating the predictivity of the test.
6-3. Chemical Coding and distribution
18.
Independent coding and distribution of chemicals were contracted out by JaCVAM to anindependent laboratory. The (company’s name) is certified according to ISO 9001, EN 4500 and GLP, andhas proven experience of reliable services. The codes were provided by JaCVAM.

7. Protocol
7-1. Protocol of the skin irritation test with LabCyte EPI-MODEL19.
In 2nd phase study, we used the SOP (ver. 5.0) and we used the SOP (ver. 6.1) in 3rd phase study.
The revised points, which make the deletion measurement of IL-1Į, revise calculating formula of viability,classification used median of 3trails and how to treat of volatile substances were shown in change trackingof the SOP (ver. 6.1). The VMT made judgments that these revise points were minor and difference with the
SOP (ver.5.0) used by 2nd phase study and this version was little in the VMT meeting on July 17, 2009.
20.
LabCyte EPI-MODEL tissues were shipped from the supplier on Mondays and delivered torecipients on Tuesdays. Upon receipt, the tissues were aseptically removed from the transport agarose
medium, transferred into 24-well plates (BD Biosciences, CA, USA) with the assay medium (0.5 mL), andincubated overnight (37°C, 5% CO2 humidified atmosphere). On the following day, the tissues weretopically exposed to the test chemicals. Liquids (25 μL) were applied with a micropipette, and solids (25mg) were applied from microtubes and moistened with 25 μL sterile water. If necessary, the mixture wasgently spread over the surface of the epidermis with a microspatula. Viscous liquids were applied using acell-saver-type tip with a micropipette. Each test chemical was applied to three tissues. In addition, threetissues serving as negative controls were treated with 25 μL distilled water, and three tissues serving as
26
46

ENV/JM/MONO(2011)39
positive controls were exposed to 5% SLS. After a 15-minute exposure, each tissue was carefully washedwith PBS (Invitrogen, CA, USA) 10 times using a washing bottle to remove any remaining test chemicalfrom the surface. The blotted tissues were then transferred to new 24-well plates containing 1 mL of fresh
assay medium.
21.
The treated and control tissues were incubated for 42 hours (37°C, 5% CO2 humidified
atmosphere). When the 42-hour post-incubation period was complete, blotted tissues were transferred to new
24-well plates containing 0.5 mL of freshly prepared MTT medium (1 mg/mL; Dojindo Co., Kumamoto,Japan) for the MTT assay and conditioned medium was collected to determine the interleukin-1 alpha (IL1Į) levels. Tissues were incubated for three hours (37°C, 5% CO2 humidified atmosphere) and thentransferred to microtubes containing 0.3 mL isopropanol, which completely immersed the tissue. Formazan
extraction was performed at room temperature, and the tissues were allowed to stand overnight.
Subsequently, 200-μL extracts were transferred to a 96-well plate. The optical density was measured at 570
nm and 650 nm as a reference absorbance, with isopropanol as a blank.
22.
The tissue viability was calculated as a percentage relative to the viability of the negative controls.
The median of three values from identically treated tissues was used to classify a chemical according to theprediction model.
23.
The amount of IL-1Į released in the conditioned medium after 42 hours was determined using an
IL-1Į ELISA kit (Invitrogen, CA, USA), following the manufacturer’s detailed instructions.

7-2. Prediction model of skin irritation
24.
In this study, the prediction model of skin irritation potential with LabCyte EPI-MODEL was set torefer to the conditions for EPISKIN described in the ECVAM Performance Standards. This prediction model
is described in Table 3. In the event that the three independent results within an individual batch were notconsistent, the result that occurred twice was used.
Acceptance criteria
1) ODNC of the negative control is greater than 0.7.
2) The viability of the positive control is less than 40%.
Table 3. Positive Criteria.
Tissue Viability (primary)
Mean tissue viability  50%
Mean tissue viability > 50%
Mean tissue viability > 50%

IL-1Į ELISA (secondary)
Mean IL-1Į release  120 pg/tissue
Mean IL-1Į release < 120 pg/tissue

Classification
Irritant
Non-irritant

7-3. Difference between LabCyte EPI-MODEL 24 protocol and EPISKIN protocol
25.
The differences between the LabCyte EPI-MODEL 24 protocol and EPISKIN protocol aresummarized in Table 3. Although the amount of medium (Table 4(A)), amount of test chemicals (Table
4(B)), and threshold of IL-1Į content (Table 4(C)) for the LabCyte EPI-MODEL 24 protocol are different
from the EPISKIN protocol, their conditions meet the descriptions of the Performance Standards.

27
47

ENV/JM/MONO(2011)39
Table 4. Differences between the LabCyte EPI-MODEL 24 protocol and EPISKIN protocol.
(A) Amount of medium.
ReasonLabCyte EPIMODEL 24
SOP

EPISKIN
SOP

Pre-incubation

0.5 mL

2 mL

Post-incubation

1 mL

2 mL

MTT assay

0.5 mL

2 mL

LabCyte EPI-MODEL 24 cultures are performed in 24-wellculture plates. A medium volume of 0.5 mL to 1 mL isappropriate to add to the 24-well culture plate. A medium
volume of 1 mL is necessary for a 42-hour culture.

These conditions meet the descriptions of the Performance Standards.
(B) Amount of test chemicals.
Test chemical

LabCyte EPI-MODEL 24
SOP

EPISKIN
SOP

ReasonLiquid

25 μL(75 μL/cm2)

10 μL(25 μL/cm2)

The lowest amount of the test chemical thatspread uniformly was applied to the test model.

Solid

25 mg+25 μL DW(75 μL/cm2)

10 mg+10 μL DW(25 μL/cm2)

These conditions meet the descriptions of the Performance Standards.
(C) Amount of test chemicals.
LabCyte EPI-MODEL 24 SOPEPISKIN SOP

Performance Standards(EPISKIN)

IL-1Į content t 120 pg/tissue(IL-1Į content t 120 pg/mL)

IL-1Į content t 100 pg/tissue(IL-1Įcontent t 50 pg/mL)

IL-1Į content t 120 pg/tissue(IL-1Į t 60 pg/mL)

The threshold of IL-1Į released in LabCyte EPI-MODEL was set based on the conditions for EPISKIN
described in the Performance Standards.
7-4. Data collection, handling, and analysis
26.
The independent biostatisticians for the study collected and organised the data using specific datacollection software (Datasheet4.0:20080910.xls in 2nd phase study and Datasheet5.0:20090430.xls in 3rdphase study). They will work in close collaboration with the biostatisticians, (Takashi Omori, Etsuyoshi
Miyaoka, and Kenya Ishiyama). After decoding the data, they will perform statistical analyses. The data
management procedures and statistical tools applied will be approved by the VMT.
7-5. Quality assurance, GLP
LABORATORIES
27.
All participating laboratories worked in the spirit of OECD GLP-like principles.
QA aspects
28.

Takashi Omori, Kenya Ishiyama and Hajime Kojima assured the quality of all the data and records.
28

48

ENV/JM/MONO(2011)39

8. Results
8-1 1st Phase
8-1-1 Negative control29.
In 1st phase data, Table 5 shows the absorbance values for the negative control. All data for thenegative control met the acceptance criteria.
Table 5. Absorbance of negative control by 1st phase study.
䎃
䎃
Lab.
a
b
c
d
e
f
g

1
Value
1.073
0.93
0.96
0.987
0.84
1.049
1.147

Exp.
2
Value
0.928
1.245
0.869
0.928
0.884
0.934
1.159

3
Value
1.007
1.042
0.761
0.939
0.973
0.968
1.074

䎃
䎃
Mean
1.003
1.072
0.863
0.951
0.899
0.984
1.127

SD
0.073
0.16
0.1
0.031
0.068
0.059
0.046

8-1-2 Positive control and test chemicals
30.
Table 6 shows the testing chemicals did not show any great score when the scores on tests wererepeated in each laboratory. Furthermore, there was no significant inter-laboratory variation. Theseexperiments suggested the feasibility of the LabCyte EPI-MODEL24 through the experiment. Alllaboratories were judged to participate at the Phase II by the validation management team.

29
49

ENV/JM/MONO(2011)39
Table 6. Viability of the positive control and three coded chemicals by 1st phase study
䎃
1
2
3
䎃
Chem. Lab. Viability Viability Viability Mean
SD
PC
a
6.35
27.55
15.67
16.52 10.63
䎃
b
3.94
3.51
3.97
3.81
0.26
䎃
c
5.45
4.81
3.49
4.58
1
䎃
d
11.74
7.22
14.08
11.02
3.49
䎃
e
31.6
9.76
38.61
26.66 15.05
䎃
f
3.1
2.89
2.93
2.97
0.11
䎃
g
4.46
7.17
2.62
4.75
2.29
P01
a
62.67
39.12
46.61
49.46 12.03
Ethanol b
41.08
50.86
86.58
59.51 23.95
䎃
c
68.13
34.13
67.31
56.53
19.4
䎃
d
68.57
40.52
33.03
47.37 18.73
䎃
e
54.19
72.08
60.55
62.27
9.07
䎃
f
.
64.16
47.98
56.07 11.44
䎃
g
4.68
5.23
6.67
5.53
1.03
P02
a
103.63104.1798.48 102.09
3.14
Glycerol b
85.5
100.5867.97
84.68 16.32
䎃c101.2499.41
104.84 101.83
2.76
䎃
d
103.3
101.35
89.73
98.13
7.34
䎃
e
101.75
98.06
99.04
99.62
1.91
䎃
f
.
97.23
96
96.62
0.87
䎃
g
94
98.16
103.6
98.59
4.82
P03
a
109.13
90.73
97.78
99.22
9.28
naphtalen b
93.96
103.91103.96 100.61
5.76
acetic acid

䎃
䎃
䎃
䎃
䎃

c
d
e
f
g

103.66102.28107.11.
92.2

102.11
98.15
104.39101.34101.04

117.3
94.56
97.36
102.07105.52

107.6998.33
102.95
101.7
99.59

8.36
3.86
5.03
0.52
6.78

8-2. 2nd phase & 3rd phase
8-2-1. Comments at the Datasheet
31.
All tests were sufficient with acceptance criteria.
There were a few comments from eachlaboratory in Tables 7 -9. By an application of Potassium hydroxide (5%aq) (B175, D213 and F232), themodel's layers were desquamated. By an application of cinnamicaldehyde (D216 and G256), the cups werediscoloured and crystallized.

30
50

ENV/JM/MONO(2011)39
Comments on the datasheets (Viability) by 2nd phase
Date
Comments
Lot
Date
Comments
Lab䇭ID Exp.No.
LCE24-081013-B
LCE24-081013-B
2008/10/20
2008/10/20aMain-䋲
This
test
was
recorded as the Main-1.
Main-2This test
recorded
LEC24-081117-B
LEC24-081117-B
2008/11/1
2008/11/1 This
This test
test was recorded
recorded as the Main-2.
Main-䋳
aMain-3
LCE24-081117-B 2008/11/22
2008/11/22 ThisaThis test
test was recorded
recorded as the Main-3.
Main-䋴
Main-4 LCE24-081117-B
LCE24-081013-B
LCE24-081013-B
2008/10/20
2008/10/20b
Main-1
LCE24-081027-B 2008.11.04
2008.11.04b
Main-2 LCE24-081027-B
LCE24-081117-B 2008/11/25
b
Main-3 LCE24-081117-B
2008/11/25
LCE24-080929-B
LCE24-080929-B
2008.10.6
c1
2008.10.6
LCE24-081020-B
LCE24-081020-B 2008/10/27
c2
2008/10/27
LCE24-081027-B
LCE24-081027-B
2008.11.3
c3
2008.11.3
LCE24-081020-B
LCE24-081020-B 2008/10/27
d81021
2008/10/27
2008/11/4
LCE24-081027-B
d81028 LCE24-081027-B
2008/11/4
LCE24-081117-B
LCE24-081117-B 2008/11/25
d81118
2008/11/25
LCE24-081006-B 2008/10/14e
Main-1 LCE24-081006-B
2008/10/14e
Main-2 LCE24-081013-B
LCE24-081013-B 2008/10/20
2008/10/20eMain-3 LCE24-081020-B
LCE24-081020-B 2008/10/27
2008/10/27
LCE24-080929-B
2008/10/6
LAB-08VAL
ff
LCE24-080929-B
LAB-08VAL
2008/10/6
LCE24-081013-B
2008/10/20
ff
MaruishiMaruishi LCE24-081013-B 2008/10/20
LAB-08VAL
ff
LCE24-081103-B 2008/11/10
LAB-08VAL LCE24-081103-B
2008/11/10
By
By anan application
application ofof G49,G53,G55,
gg
Main-1
LCE24-080929-B 2008.10.06
2008.10.06
Main-1 LCE24-080929-B
themodel's cap thewasmodel'sdiscolored.
G49,G53,G55,
cap wasByanapplication
ofG49,G53,G55,
Byan
applicationof
LCE24-081013-B
gg
Main-2
2008.10.20 the model's cap was discolored.
Main-2 LCE24-081013-B 2008.10.20
G49,G53,G55, the model's cap wasBy
By anan application
application ofof G49,G53,G55,
LCE24-081027-B
2008.11.03
gg
Main-3Main-3 LCE24-081027-B 2008.11.03 the model's cap was discolored.
G49,G53,G55, the model's cap was

Table 7.

Table 8. Comments on the datasheets (ELISA) by 2nd phase
Lab_ID
Lot
Date
Comments
Lab_ID Exp.No.
Exp.No.
Lot
DateComments
LCE24-081013-B
2008/10/20
LCE24-081013-B
2008/10/20 Thisaa
MainMain-2
This test
test was
was recorded
recordedas
asthe
the Main-1.
䋲
LEC24-081117-B
LEC24-081117-B
2008/11/1
2008/11/1 Thisaa
MainMain-3
䋳This test
test was
was recorded
recordedas
asthe
the Main-2.
LCE24-081117-B
LCE24-081117-B
2008/11/22
2008/11/22 Thisaa
MainMain-4
This test
test was
was recorded
recordedas
asthe
the Main-3.
䋴
LCE24-081013-B
LCE24-081013-B 2008/12/12
2008/12/12
Main-1bb
Main-1
LCE24-081027-B 2008/12/12
LCE24-081027-B
Main-2
2008/12/12
bb
Main-2
LCE24-081117-B 2008.12.26
LCE24-081117-B
2008.12.26
Main-3bb
Main-3
LCE24-080929-B
LCE24-080929-B
2008/10/7
cc
11
2008/10/7
LCE24-081020-B
LCE24-081020-B
2008/10/30
cc22
2008/10/30
LCE24-081027-B 2008.11.3
LCE24-081027-B
2008.11.3
cc
33
LCE24-081020-B 2008/11/11
LCE24-081020-B
2008/11/11
81021
dd
81021
LCE24-081027-B
2008/11/26
81028
LCE24-081027-B 2008/11/26
dd
81028
LCE24-081117-B
81118
2009/1/7
LCE24-081117-B
2009/1/7
dd
81118
LCE24-081006-B
2008/12/2eMain-1
LCE24-081006-B
e
Main-1
2008/12/2
LCE24-081013-B
2008/12/2
Main-2
LCE24-081013-B
ee
Main-2
2008/12/2
LCE24-081020-B
e
Main-3
2008/12/19
LCE24-081020-B
e
Main-3
2008/12/19
LCE24-081013-B 2008/11/25
2008/11/25
Maruishi LCE24-081013-B
ff
Maruishi
LCE24-081013-B
2008/11/27
f
Maruishi
LCE24-081013-B
f
Maruishi2008/11/27
LCE24-081103-B
f
LAB-08VAL
2008/12/25
f
LAB-08VAL LCE24-081103-B 2008/12/25
LCE24-080929-B 2008.10.09
2008.10.09
Main-1
LCE24-080929-B
gg
Main-1
LCE24-081013-B
2008.10.22
gMain-2
LCE24-081013-B 2008.10.22
gMain-2
LCE24-081027-B
g
Main-32008.11.05
LCE24-081027-B 2008.11.05
gMain-331
51

ENV/JM/MONO(2011)39
Table 9. Comments on the datasheets (Viability) by 3rd phase study
Lab䇭ID

a
a
a
b
b
bb
cc
cc
cc
dd
d

d
d
f
ff
f
f
f
g
g
g
g
g
g

Exp.No.
Lot
Date
Date
Comments
Comments
LCE24-090420-A
LCE24-090420-A 2009/4/27
㪥㫆㪅㪈
2009/4/27
LEC24-090511-A
LEC24-090511-A 2009/5/18
㪥㫆㪅㪉
2009/5/18
LEC24-090518-A
LEC24-090518-A 2009/5/25
㪥㫆㪅㪊
2009/5/25
20090421㪄㪈 LCE24-090420-A
LCE24-090420-A 2009/4/27
2009/4/27 By
By an
an application
applicationof
ofB175,
B175,thethemodel'smodel'slayers
layerswere
weredesquamated䋮
desquamated䋮
20090421㪄㪉 LEC24-090511-A
LEC24-090511-A 2009/5/20
2009/5/20 By
By an
an application
applicationof
ofB175,
B175,thethemodel'smodel'slayers
layerswere
weredesquamated䋮
desquamated䋮
20090421㪄㪊
LEC24-090518-A 2009/5/25
20090421㪄㪊 LEC24-090518-A
2009/5/25 By
By an
an application
applicationof
ofB175,
B175,thethemodel'smodel'slayers
layerswere
weredesquamated䋮
desquamated䋮
LCE24-090420-A
11
LCE24-090420-A 2009/4/27
2009/4/27
LEC24-090511-A
22
LEC24-090511-A 2009/5/18
2009/5/18
LEC24-090518-A 2009/5/25
33
LEC24-090518-A 2009/5/25
By an
an application
application ofofD213,
D213,thethemodel'smodel'slayers
layerswere
weredesquamated䋮
desquamated䋮By
By
By
LEC24-090511-A 2009/5/18
90512
LEC24-090511-A 2009/5/18 an application of D216, white crystallizations in the cup were90512
an application of D216, white crystallizations in the cup were detected.
detected.
By an application of D213, the model's layers were desquamated䋮 By
By
an application of D213, the model's layers were desquamated䋮 By
LEC24-090518-A 2009/5/25
LEC24-090518-A
2009/5/25 an application of D216, white crystallizations in the cup were90519
an application of D216, white crystallizations in the cup were detected.
detected
By an application of D213, the model's layers were desquamated䋮 By
LEC24-090525-A
90526
2009/6/1 By
an applicationof D216,
whitein thecup werean applicationof D213,the crystallizations
model's layers were
desquamated䋮
By
LEC24-090525-A
2009/6/1
90526
detected
an application of D216, white crystallizations in the cup were detected.
LAB-09VAL LCE24-090420-A 2009/4/27 By an application of F232, the model's layers were desquamated䋮
LCE24-090420-A
LAB-09VAL
LAB-09VAL LEC24-090511-A 2009/4/27
2009/5/18 By an application of F232, the model's layers were desquamated䋮
LEC24-090511-A
LAB-09VAL
2009/5/18
LAB-09VAL LEC24-090518-A 2009/5/25 By an application of F232, the model's layers were desquamated䋮
LAB-09VAL LEC24-090518-A 2009/5/25 By an application of F232, the model's layers were desquamated䋮
LCE24-090420-A 2009/4/27 By an application of G256, the model's caps were discolored䋮㽲
LCE24-090420-A 2009/4/27 By an application of G256, the model's caps were discolored䋮㽲
LCE24-090427-A
2009/5/4 By an application of G256, the model's caps were discolored䋮㽳
LCE24-090427-A
2009/5/4 By
㽳
LEC24-090511-A 2009/5/18
By an
an application
applicationof
ofG256,
G256,thethemodel'smodel'scaps
capswere
werediscolored䋮
discolored䋮㽴
LEC24-090511-A 2009/5/18 By an application of G256, the model's caps were discolored䋮㽴

8-2-2. Negative control
32.
In Table 10 and Fig.2, absorbances of negative control are shown. All data of negative control
were sufficient with acceptance criteria excluding Lab a, test1. The mean OD of lab a, test 1 is 0.59 (0.61,
0.58, 0.57). We were not accepted at this result, and accepted the results of test 2-4 re-tested at Lab a.

32
52

ENV/JM/MONO(2011)39
Table 10㩷 Absorbance of negative control

2
3

OD
Fig.2 Distribution of Absorbance on negative control
8-2-3. Positive control
33.
Table 11 and Fig.3 show three independent viabilities and statistical analysis of positive control ateach laboratory. All data were sufficient with acceptance criteria of positive control.

33
53

ENV/JM/MONO(2011)39

Table 11. Viability of positive control

2
3

Fig.3 Distribution of viability on positive control
8-2-4. Skin irritation test by cell viability
34.
The results of the skin irritation test with LabCyte EPI-MODEL 24 when it was only evaluated cell
viabilities as indicator are shown in Table 12 in 2nd phase study and Table 14 in 3rd phase study. Summary㩷statistical analysis of viability each chemical are shown in Table 13 and Fig.4 in 2nd phase study and Table
15 and Fig.5 in 3rd phase study.
35.
Invalid data obtained only Lab a, run 1. This lab performed at retesting. Therefore, the data of lab
a were accepted among run 2-4.

34
54

ENV/JM/MONO(2011)39

Table 12. Viability of chemicals at each laboratory by 2nd phase study.
䎦䏋䏈䏐䎑 䎹䏌䏙䏒 䎶䏆䏒䏕䏈 䎨䏛䏓䎑

䎓䎔

䏑䏒

䎓

䎓䎕

䏑䏒

䎓

䎓䎖

䏑䏒

䎓

䎓䎗

䏑䏒

䎓

䎓䎘

䏑䏒

䎓䎑䎖

䎓䎙

䏑䏒

䎓䎑䎖

䎓䎚

䏑䏒

䎔

䎓䎛

䏑䏒

䎔

䎓䎜

䏑䏒

䎔䎑䎚

䎔䎓

䏑䏒

䎔䎑䎚

䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖

䏄
䎖䎔䎑䎓
䎔䎔䎑䎕
䎔䎔䎑䎙
䎚䎜䎑䎛
䎚䎙䎑䎘
䎙䎘䎑䎕
䎔䎓䎜䎑䎔
䎔䎓䎖䎑䎜
䎔䎓䎓䎑䎜
䎔䎓䎙䎑䎖
䎜䎘䎑䎕
䎜䎙䎑䎘
䎚䎛䎑䎘
䎚䎛䎑䎘
䎚䎗䎑䎔
䎜䎕䎑䎘
䎚䎜䎑䎗
䎛䎕䎑䎗
䎕䎗䎑䎔
䎔䎕䎑䎙
䎔䎚䎑䎛
䎔䎔䎔䎑䎜
䎜䎓䎑䎕
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䎔䎓䎔䎑䎔
䎔䎔䎘䎑䎜
䎔䎓䎗䎑䎔
䎛䎙䎑䎘

䏅
䎗䎚䎑䎔
䎔䎓䎑䎗
䎔䎙䎑䎔
䎙䎙䎑䎜
䎙䎔䎑䎚
䎛䎛䎑䎚
䎜䎖䎑䎖
䎜䎜䎑䎛
䎔䎓䎕䎑䎖
䎜䎗䎑䎗
䎔䎓䎓䎑䎕
䎜䎛䎑䎙
䎙䎔䎑䎚
䎚䎔䎑䎜
䎛䎗䎑䎘
䎚䎚䎑䎜
䎛䎖䎑䎘
䎛䎓䎑䎘
䎔䎓䎑䎛
䎔䎕䎑䎙
䎔䎖䎑䎕
䎛䎙䎑䎚
䎔䎓䎓䎑䎙
䎔䎓䎗䎑䎛
䎜䎙䎑䎚
䎔䎔䎓䎑䎔
䎔䎓䎜䎑䎘
䎔䎔䎘䎑䎗
䎔䎔䎓䎑䎔
䎔䎔䎔䎑䎖

䏆
䎔䎓䎑䎙
䎕䎓䎑䎖
䎔䎕䎑䎗
䎛䎛䎑䎔
䎛䎜䎑䎚
䎛䎘䎑䎛
䎜䎗䎑䎙
䎜䎖䎑䎔
䎜䎘䎑䎚
䎜䎚䎑䎔
䎜䎜䎑䎜
䎜䎚䎑䎛
䎜䎔䎑䎗
䎜䎘䎑䎕
䎛䎜䎑䎕
䎛䎔䎑䎓
䎚䎜䎑䎔
䎛䎖䎑䎙
䎕䎓䎑䎛
䎔䎙䎑䎕
䎔䎘䎑䎕
䎚䎘䎑䎖
䎛䎕䎑䎖
䎚䎚䎑䎕
䎔䎓䎙䎑䎙
䎜䎙䎑䎛
䎜䎖䎑䎘
䎔䎓䎚䎑䎘
䎔䎓䎖䎑䎙
䎔䎓䎖䎑䎚

䎯䏄䏅䎑
䏇
䎔䎗䎑䎖
䎜䎑䎔
䎜䎑䎙
䎔䎓䎕䎑䎖
䎛䎜䎑䎛
䎙䎚䎑䎙
䎔䎓䎘䎑䎔
䎔䎔䎕䎑䎛
䎔䎓䎔䎑䎗
䎔䎓䎙䎑䎔
䎔䎓䎓䎑䎜
䎜䎛䎑䎗
䎚䎜䎑䎗
䎚䎓䎑䎘
䎙䎙䎑䎔
䎜䎔䎑䎖
䎔䎓䎕䎑䎗
䎛䎕䎑䎚
䎕䎔䎑䎚
䎔䎖䎑䎛
䎔䎜䎑䎛
䎔䎓䎜䎑䎗
䎔䎓䎚䎑䎘
䎔䎓䎖䎑䎓
䎔䎓䎘䎑䎓
䎔䎓䎖䎑䎗
䎜䎛䎑䎔
䎔䎔䎗䎑䎖
䎔䎓䎛䎑䎕
䎔䎓䎘䎑䎘

䏈
䎖䎛䎑䎔
䎕䎘䎑䎕
䎖䎕䎑䎖
䎔䎓䎔䎑䎛
䎚䎙䎑䎗
䎛䎘䎑䎛
䎔䎕䎜䎑䎙
䎔䎓䎙䎑䎙
䎔䎓䎖䎑䎜
䎔䎕䎚䎑䎔
䎔䎔䎖䎑䎙
䎔䎓䎘䎑䎕
䎔䎓䎖䎑䎓
䎜䎓䎑䎖
䎛䎜䎑䎙
䎜䎚䎑䎓
䎛䎔䎑䎘
䎜䎓䎑䎚
䎔䎚䎑䎘
䎕䎕䎑䎕
䎕䎔䎑䎖
䎔䎔䎗䎑䎜
䎔䎓䎓䎑䎜
䎔䎓䎓䎑䎜
䎔䎔䎘䎑䎛
䎔䎓䎛䎑䎙
䎔䎓䎖䎑䎜
䎔䎖䎕䎑䎓
䎔䎔䎚䎑䎓
䎔䎓䎚䎑䎘

䏉
䎔䎗䎑䎖
䎔䎔䎑䎕
䎔䎓䎑䎗
䎚䎘䎑䎖
䎙䎚䎑䎕
䎚䎘䎑䎚
䎜䎗䎑䎕
䎜䎚䎑䎜
䎜䎕䎑䎘
䎔䎓䎓䎑䎔
䎜䎕䎑䎛
䎜䎕䎑䎚
䎚䎔䎑䎜
䎖䎜䎑䎖
䎘䎘䎑䎔
䎛䎚䎑䎛
䎜䎗䎑䎗
䎛䎔䎑䎔
䎔䎘䎑䎛
䎖䎔䎑䎔
䎔䎘䎑䎙
䎛䎜䎑䎚
䎜䎚䎑䎛
䎜䎙䎑䎘
䎜䎛䎑䎛
䎛䎙䎑䎘
䎜䎚䎑䎚
䎔䎓䎗䎑䎓
䎔䎓䎔䎑䎕
䎔䎓䎔䎑䎕

䏊
䎔䎓䎑䎙
䎔䎓䎑䎙
䎔䎗䎑䎓
䎜䎙䎑䎓
䎜䎗䎑䎛
䎔䎓䎖䎑䎖
䎔䎓䎓䎑䎘
䎜䎖䎑䎗
䎔䎔䎔䎑䎔
䎔䎓䎗䎑䎛
䎔䎓䎖䎑䎖
䎔䎓䎜䎑䎛
䎜䎙䎑䎛
䎛䎜䎑䎜
䎛䎛䎑䎗
䎛䎚䎑䎕
䎛䎔䎑䎕
䎘䎗䎑䎔
䎖䎔䎑䎘
䎕䎕䎑䎘
䎔䎜䎑䎜
䎔䎓䎔䎑䎔
䎔䎓䎓䎑䎜
䎔䎓䎜䎑䎓
䎔䎓䎕䎑䎖
䎔䎓䎖䎑䎗
䎔䎔䎕䎑䎔
䎔䎓䎚䎑䎜
䎔䎓䎛䎑䎗
䎔䎔䎖䎑䎔

35
55

ENV/JM/MONO(2011)39
Table 12.

continued

䎦䏋䏈䏐䎑

䎹䏌䏙䏒

䎔䎔

䏑䏒

䎕

䎔䎕

䏑䏒

䎕

䎶䏆䏒䏕䏈 䎨䏛䏓䎑䎔䎗䎦䏄䏗䏈䏊䏒䏕䏜䎕䎑䎖䎕䎔䎘䎦䏄䏗䏈䏊䏒䏕䏜䎕䎑䎖䎕䎔䎙䎦䏄䏗䏈䏊䏒䏕䏜䎕䎑䎚䎕䎔䎚䎦䏄䏗䏈䏊䏒䏕䏜䎕䎑䎚䎕䎔䎛䎦䏄䏗䏈䏊䏒䏕䏜䎕䎖䎔䎜䎦䏄䏗䏈䏊䏒䏕䏜䎕䎖䎕䎓䎦䏄䏗䏈䏊䏒䏕䏜
䎕

䎗

䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖

䏄
䎔䎔䎖䎑䎚
䎜䎛䎑䎔
䎔䎔䎕䎑䎙
䎕䎛䎑䎕
䎔䎛䎑䎗
䎔䎘䎑䎖
䎔䎔䎑䎔
䎙䎑䎙
䎙䎑䎛
䎔䎔䎑䎔
䎚䎑䎔
䎛䎑䎕
䎙䎚䎑䎜
䎖䎕䎑䎕
䎘䎜䎑䎛
䎙䎑䎔
䎗䎑䎛
䎘䎑䎙
䎛䎕䎑䎔
䎚䎛䎑䎖
䎕䎘䎑䎖
䎔䎘䎑䎓
䎔䎜䎑䎜
䎘䎔䎑䎔
䎖䎔䎑䎔
䎜䎑䎖
䎕䎜䎑䎘

䎯䏄䏅䎑
䏅
䏆
䏇
䏈
䏉
䏊
䎔䎓䎘䎑䎓 䎔䎓䎔䎑䎓 䎔䎓䎕䎑䎗 䎔䎕䎖䎑䎔 䎔䎓䎖䎑䎔 䎔䎓䎕䎑䎛䎔䎓䎙䎑䎙 䎜䎗䎑䎙 䎔䎓䎘䎑䎛 䎔䎔䎓䎑䎗 䎜䎛䎑䎓 䎔䎓䎓䎑䎘䎔䎓䎖䎑䎚 䎜䎗䎑䎔 䎔䎓䎕䎑䎚 䎔䎓䎘䎑䎘 䎜䎗䎑䎙 䎔䎓䎜䎑䎓䎕䎗䎑䎙 䎕䎗䎑䎜 䎘䎗䎑䎖 䎘䎘䎑䎙 䎕䎚䎑䎕 䎛䎚䎑䎚䎕䎗䎑䎙 䎗䎗䎑䎛 䎚䎙䎑䎕 䎘䎚䎑䎛 䎙䎘䎑䎕 䎜䎛䎑䎓䎔䎘䎑䎜 䎕䎛䎑䎔 䎕䎚䎑䎗 䎘䎚䎑䎕 䎙䎙䎑䎓 䎔䎔䎕䎑䎙䎔䎕䎑䎔 䎔䎗䎑䎚 䎔䎓䎑䎚 䎔䎗䎑䎕 䎔䎖䎑䎔 䎔䎖䎑䎘䎛䎑䎖䎜䎑䎘 䎔䎔䎑䎚 䎔䎕䎑䎓 䎔䎙䎑䎚 䎔䎕䎑䎓䎛䎑䎛䎜䎑䎔 䎔䎓䎑䎕 䎔䎓䎑䎗 䎔䎚䎑䎓 䎔䎓䎑䎙䎜䎑䎖 䎔䎖䎑䎔䎛䎑䎓 䎔䎔䎑䎓
䎛䎑䎙䎜䎑䎕䎔䎓䎑䎕 䎔䎜䎑䎖䎛䎑䎙 䎔䎔䎑䎖䎘䎑䎜 䎕䎗䎑䎚
䎜䎑䎜
䎛䎑䎔
䎜䎑䎕
䎛䎑䎚
䎚䎑䎔
䎜䎑䎕
䎜䎕䎑䎓 䎘䎔䎑䎘 䎔䎛䎑䎔 䎜䎛䎑䎕 䎘䎜䎑䎙 䎙䎗䎑䎜䎘䎗䎑䎔 䎛䎙䎑䎖 䎚䎜䎑䎕 䎜䎓䎑䎙 䎘䎓䎑䎗 䎚䎜䎑䎙䎜䎛䎑䎖 䎛䎔䎑䎚 䎖䎚䎑䎚 䎚䎛䎑䎚 䎙䎚䎑䎘 䎛䎙䎑䎘
䎗䎑䎘
䎘䎑䎖
䎙䎑䎙
䎛䎑䎜
䎙䎑䎜
䎙䎑䎕
䎗䎑䎚
䎙䎑䎓
䎘䎑䎖
䎙䎑䎖
䎘䎑䎘
䎘䎑䎖
䎘䎑䎚
䎘䎑䎜
䎖䎑䎜
䎘䎑䎗
䎗䎑䎘
䎘䎑䎖
䎗䎙䎑䎘 䎜䎔䎑䎕 䎛䎖䎑䎚 䎜䎛䎑䎜 䎙䎜䎑䎕 䎜䎕䎑䎗䎘䎓䎑䎙 䎛䎚䎑䎖 䎙䎜䎑䎜 䎛䎚䎑䎕 䎛䎓䎑䎙 䎛䎘䎑䎜䎔䎓䎓䎑䎓 䎛䎚䎑䎘 䎘䎜䎑䎓 䎙䎜䎑䎔 䎚䎔䎑䎜 䎜䎗䎑䎗䎚䎗䎑䎙 䎔䎓䎑䎓 䎖䎓䎑䎗 䎛䎖䎑䎔 䎗䎓䎑䎔 䎖䎘䎑䎛䎔䎓䎑䎜 䎕䎕䎑䎗 䎕䎛䎑䎖 䎕䎙䎑䎔 䎛䎚䎑䎓 䎗䎗䎑䎚䎖䎕䎑䎓 䎖䎘䎑䎓 䎔䎛䎑䎕 䎙䎜䎑䎗 䎚䎔䎑䎛 䎖䎛䎑䎚䎕䎗䎑䎛 䎔䎓䎑䎗䎜䎑䎙 䎔䎓䎑䎚
䎛䎑䎔
䎛䎑䎛
䎛䎑䎓
䎚䎑䎙 䎔䎙䎑䎜
䎛䎑䎕
䎚䎑䎛
䎙䎑䎚
䎜䎑䎖
䎚䎑䎙 䎖䎓䎑䎜
䎙䎑䎕
䎛䎑䎕
䎛䎑䎙

36
56

ENV/JM/MONO(2011)39
Table 13. Summary of the statistical analysis of the viability for each chemical by 2nd phase study.
䎦䏋䏈䏐䎑 䎶䏗䏄䏗䎑䎰䏈䏄䏑䎰䏈䏇䏌䏄䏑

䎓䎔

䎓䎕

䎓䎖

䎓䎗

䎓䎘

䎓䎙

䎓䎚

䎓䎛

䎓䎜

䎔䎓

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛

䏄
䎔䎚䎑䎜
䎔䎔䎑䎙
䎔䎔䎑䎕
䎖䎔䎑䎓
䎚䎖䎑䎛
䎚䎙䎑䎘
䎙䎘䎑䎕
䎚䎜䎑䎛
䎔䎓䎗䎑䎚
䎔䎓䎖䎑䎜
䎔䎓䎓䎑䎜
䎔䎓䎜䎑䎔
䎜䎜䎑䎖
䎜䎙䎑䎘
䎜䎘䎑䎕
䎔䎓䎙䎑䎖
䎚䎚䎑䎓
䎚䎛䎑䎘
䎚䎗䎑䎔
䎚䎛䎑䎘
䎛䎗䎑䎛
䎛䎕䎑䎗
䎚䎜䎑䎗
䎜䎕䎑䎘
䎔䎛䎑䎕
䎔䎚䎑䎛
䎔䎕䎑䎙
䎕䎗䎑䎔
䎜䎜䎑䎔
䎜䎘䎑䎖
䎜䎓䎑䎕
䎔䎔䎔䎑䎜
䎔䎓䎖䎑䎚
䎔䎓䎔䎑䎔
䎜䎚䎑䎔
䎔䎔䎕䎑䎛
䎔䎓䎕䎑䎔
䎔䎓䎗䎑䎔
䎛䎙䎑䎘
䎔䎔䎘䎑䎜

䏅
䎕䎗䎑䎘
䎔䎙䎑䎔
䎔䎓䎑䎗
䎗䎚䎑䎔
䎚䎕䎑䎗
䎙䎙䎑䎜
䎙䎔䎑䎚
䎛䎛䎑䎚
䎜䎛䎑䎘
䎜䎜䎑䎛
䎜䎖䎑䎖
䎔䎓䎕䎑䎖
䎜䎚䎑䎛
䎜䎛䎑䎙
䎜䎗䎑䎗
䎔䎓䎓䎑䎕
䎚䎕䎑䎚
䎚䎔䎑䎜
䎙䎔䎑䎚
䎛䎗䎑䎘
䎛䎓䎑䎚
䎛䎓䎑䎘
䎚䎚䎑䎜
䎛䎖䎑䎘
䎔䎕䎑䎕
䎔䎕䎑䎙
䎔䎓䎑䎛
䎔䎖䎑䎕
䎜䎚䎑䎗
䎔䎓䎓䎑䎙
䎛䎙䎑䎚
䎔䎓䎗䎑䎛
䎔䎓䎘䎑䎗
䎔䎓䎜䎑䎘
䎜䎙䎑䎚
䎔䎔䎓䎑䎔
䎔䎔䎕䎑䎕
䎔䎔䎔䎑䎖
䎔䎔䎓䎑䎔
䎔䎔䎘䎑䎗

䏆
䎔䎗䎑䎗
䎔䎕䎑䎗
䎔䎓䎑䎙
䎕䎓䎑䎖
䎛䎚䎑䎛
䎛䎛䎑䎔
䎛䎘䎑䎛
䎛䎜䎑䎚
䎜䎗䎑䎘
䎜䎗䎑䎙
䎜䎖䎑䎔
䎜䎘䎑䎚
䎜䎛䎑䎕
䎜䎚䎑䎛
䎜䎚䎑䎔
䎜䎜䎑䎜
䎜䎔䎑䎜
䎜䎔䎑䎗
䎛䎜䎑䎕
䎜䎘䎑䎕
䎛䎔䎑䎕
䎛䎔䎑䎓
䎚䎜䎑䎔
䎛䎖䎑䎙
䎔䎚䎑䎗
䎔䎙䎑䎕
䎔䎘䎑䎕
䎕䎓䎑䎛
䎚䎛䎑䎖
䎚䎚䎑䎕
䎚䎘䎑䎖
䎛䎕䎑䎖
䎜䎛䎑䎜
䎜䎙䎑䎛
䎜䎖䎑䎘
䎔䎓䎙䎑䎙
䎔䎓䎗䎑䎜
䎔䎓䎖䎑䎚
䎔䎓䎖䎑䎙
䎔䎓䎚䎑䎘

䎯䏄䏅䎑
䏇
䎔䎔䎑䎓
䎜䎑䎙
䎜䎑䎔
䎔䎗䎑䎖
䎛䎙䎑䎙
䎛䎜䎑䎛
䎙䎚䎑䎙
䎔䎓䎕䎑䎖
䎔䎓䎙䎑䎗
䎔䎓䎘䎑䎔
䎔䎓䎔䎑䎗
䎔䎔䎕䎑䎛
䎔䎓䎔䎑䎛
䎔䎓䎓䎑䎜
䎜䎛䎑䎗
䎔䎓䎙䎑䎔
䎚䎕䎑䎓
䎚䎓䎑䎘
䎙䎙䎑䎔
䎚䎜䎑䎗
䎜䎕䎑䎔
䎜䎔䎑䎖
䎛䎕䎑䎚
䎔䎓䎕䎑䎗
䎔䎛䎑䎗
䎔䎜䎑䎛
䎔䎖䎑䎛
䎕䎔䎑䎚
䎔䎓䎙䎑䎙
䎔䎓䎚䎑䎘
䎔䎓䎖䎑䎓
䎔䎓䎜䎑䎗
䎔䎓䎕䎑䎕
䎔䎓䎖䎑䎗
䎜䎛䎑䎔
䎔䎓䎘䎑䎓
䎔䎓䎜䎑䎖
䎔䎓䎛䎑䎕
䎔䎓䎘䎑䎘
䎔䎔䎗䎑䎖

䏈
䎖䎔䎑䎜
䎖䎕䎑䎖
䎕䎘䎑䎕
䎖䎛䎑䎔
䎛䎛䎑䎓
䎛䎘䎑䎛
䎚䎙䎑䎗
䎔䎓䎔䎑䎛
䎔䎔䎖䎑䎖
䎔䎓䎙䎑䎙
䎔䎓䎖䎑䎜
䎔䎕䎜䎑䎙
䎔䎔䎘䎑䎖
䎔䎔䎖䎑䎙
䎔䎓䎘䎑䎕
䎔䎕䎚䎑䎔
䎜䎗䎑䎖
䎜䎓䎑䎖
䎛䎜䎑䎙
䎔䎓䎖䎑䎓
䎛䎜䎑䎚
䎜䎓䎑䎚
䎛䎔䎑䎘
䎜䎚䎑䎓
䎕䎓䎑䎖
䎕䎔䎑䎖
䎔䎚䎑䎘
䎕䎕䎑䎕
䎔䎓䎘䎑䎙
䎔䎓䎓䎑䎜
䎔䎓䎓䎑䎜
䎔䎔䎗䎑䎜
䎔䎓䎜䎑䎗
䎔䎓䎛䎑䎙
䎔䎓䎖䎑䎜
䎔䎔䎘䎑䎛
䎔䎔䎛䎑䎛
䎔䎔䎚䎑䎓
䎔䎓䎚䎑䎘
䎔䎖䎕䎑䎓

䏉
䎔䎕䎑䎓
䎔䎔䎑䎕
䎔䎓䎑䎗
䎔䎗䎑䎖
䎚䎕䎑䎚
䎚䎘䎑䎖
䎙䎚䎑䎕
䎚䎘䎑䎚
䎜䎗䎑䎛
䎜䎗䎑䎕
䎜䎕䎑䎘
䎜䎚䎑䎜
䎜䎘䎑䎕
䎜䎕䎑䎛
䎜䎕䎑䎚
䎔䎓䎓䎑䎔
䎘䎘䎑䎗
䎘䎘䎑䎔
䎖䎜䎑䎖
䎚䎔䎑䎜
䎛䎚䎑䎛
䎛䎚䎑䎛
䎛䎔䎑䎔
䎜䎗䎑䎗
䎕䎓䎑䎛
䎔䎘䎑䎛
䎔䎘䎑䎙
䎖䎔䎑䎔
䎜䎗䎑䎚
䎜䎙䎑䎘
䎛䎜䎑䎚
䎜䎚䎑䎛
䎜䎗䎑䎖
䎜䎚䎑䎚
䎛䎙䎑䎘
䎜䎛䎑䎛
䎔䎓䎕䎑䎔
䎔䎓䎔䎑䎕
䎔䎓䎔䎑䎕
䎔䎓䎗䎑䎓

䏊
䎔䎔䎑䎚
䎔䎓䎑䎙
䎔䎓䎑䎙
䎔䎗䎑䎓
䎜䎛䎑䎓
䎜䎙䎑䎓
䎜䎗䎑䎛
䎔䎓䎖䎑䎖
䎔䎓䎔䎑䎚
䎔䎓䎓䎑䎘
䎜䎖䎑䎗
䎔䎔䎔䎑䎔
䎔䎓䎘䎑䎜
䎔䎓䎗䎑䎛
䎔䎓䎖䎑䎖
䎔䎓䎜䎑䎛
䎜䎔䎑䎚
䎛䎜䎑䎜
䎛䎛䎑䎗
䎜䎙䎑䎛
䎚䎗䎑䎕
䎛䎔䎑䎕
䎘䎗䎑䎔
䎛䎚䎑䎕
䎕䎗䎑䎙
䎕䎕䎑䎘
䎔䎜䎑䎜
䎖䎔䎑䎘
䎔䎓䎖䎑䎚
䎔䎓䎔䎑䎔
䎔䎓䎓䎑䎜
䎔䎓䎜䎑䎓
䎔䎓䎘䎑䎜
䎔䎓䎖䎑䎗
䎔䎓䎕䎑䎖
䎔䎔䎕䎑䎔
䎔䎓䎜䎑䎛
䎔䎓䎛䎑䎗
䎔䎓䎚䎑䎜
䎔䎔䎖䎑䎔

37
57

ENV/JM/MONO(2011)39

Table 13. continued.
䎦䏋䏈䏐䎑 䎶䏗䏄䏗䎑䏄䏅䎰䏈䏄䏑 䎔䎓䎛䎑䎔 䎔䎓䎘䎑䎔䎰䏈䏇䏌䏄䏑 䎔䎔䎕䎑䎙 䎔䎓䎘䎑䎓

䎔䎔

䎔䎕

䎔䎗

䎔䎘

䎔䎙

䎔䎚

䎔䎛

䎔䎜

䎕䎓

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑䎰䏄䏛䎜䎛䎑䎔 䎔䎓䎖䎑䎚䎔䎔䎖䎑䎚 䎔䎓䎙䎑䎙䎕䎓䎑䎚 䎕䎔䎑䎚䎔䎛䎑䎗 䎕䎗䎑䎙䎔䎘䎑䎖 䎔䎘䎑䎜䎕䎛䎑䎕 䎕䎗䎑䎙
䎛䎑䎕
䎜䎑䎚
䎙䎑䎛
䎛䎑䎛
䎙䎑䎙
䎛䎑䎖
䎔䎔䎑䎔 䎔䎕䎑䎔
䎛䎑䎛
䎜䎑䎛
䎛䎑䎕
䎜䎑䎜
䎚䎑䎔
䎜䎑䎖
䎔䎔䎑䎔 䎔䎓䎑䎕䎘䎖䎑䎖 䎛䎔䎑䎗䎘䎜䎑䎛 䎜䎕䎑䎓䎖䎕䎑䎕 䎘䎗䎑䎔䎙䎚䎑䎜 䎜䎛䎑䎖
䎘䎑䎘
䎗䎑䎜
䎘䎑䎙
䎗䎑䎚
䎗䎑䎛
䎗䎑䎘
䎙䎑䎔
䎘䎑䎚
䎙䎔䎑䎜 䎙䎘䎑䎚䎚䎛䎑䎖 䎘䎓䎑䎙䎕䎘䎑䎖 䎗䎙䎑䎘䎛䎕䎑䎔 䎔䎓䎓䎑䎓䎕䎛䎑䎚 䎖䎜䎑䎕䎔䎜䎑䎜 䎖䎕䎑䎓䎔䎘䎑䎓 䎔䎓䎑䎜䎘䎔䎑䎔 䎚䎗䎑䎙䎕䎖䎑䎖 䎔䎗䎑䎓
䎕䎜䎑䎘
䎜䎑䎖
䎜䎑䎖
䎛䎑䎓
䎖䎔䎑䎔 䎕䎗䎑䎛

䏆
䎜䎙䎑䎙
䎜䎗䎑䎙
䎜䎗䎑䎔
䎔䎓䎔䎑䎓
䎖䎕䎑䎙
䎕䎛䎑䎔
䎕䎗䎑䎜
䎗䎗䎑䎛
䎔䎔䎑䎔
䎜䎑䎘
䎜䎑䎔
䎔䎗䎑䎚
䎔䎖䎑䎘
䎔䎖䎑䎔
䎛䎑䎔
䎔䎜䎑䎖
䎚䎖䎑䎔
䎛䎔䎑䎚
䎘䎔䎑䎘
䎛䎙䎑䎖
䎘䎑䎛
䎘䎑䎜
䎘䎑䎖
䎙䎑䎓
䎛䎛䎑䎚
䎛䎚䎑䎘
䎛䎚䎑䎖
䎜䎔䎑䎕
䎕䎕䎑䎘
䎕䎕䎑䎗
䎔䎓䎑䎓
䎖䎘䎑䎓
䎛䎑䎙
䎚䎑䎙
䎚䎑䎙
䎔䎓䎑䎗

䎯䏄䏅䎑
䏇
䎔䎓䎖䎑䎙
䎔䎓䎕䎑䎚
䎔䎓䎕䎑䎗
䎔䎓䎘䎑䎛
䎘䎕䎑䎙
䎘䎗䎑䎖
䎕䎚䎑䎗
䎚䎙䎑䎕
䎔䎓䎑䎜
䎔䎓䎑䎚
䎔䎓䎑䎕
䎔䎔䎑䎚
䎛䎑䎙
䎛䎑䎙
䎛䎑䎓
䎜䎑䎕
䎗䎘䎑䎓
䎖䎚䎑䎚
䎔䎛䎑䎔
䎚䎜䎑䎕
䎘䎑䎖
䎘䎑䎖
䎖䎑䎜
䎙䎑䎙
䎚䎓䎑䎜
䎙䎜䎑䎜
䎘䎜䎑䎓
䎛䎖䎑䎚
䎕䎘䎑䎙
䎕䎛䎑䎖
䎔䎛䎑䎕
䎖䎓䎑䎗
䎔䎜䎑䎕
䎔䎙䎑䎜
䎜䎑䎙
䎖䎓䎑䎜

䏈
䏉
䏊
䎔䎔䎖䎑䎓 䎜䎛䎑䎙 䎔䎓䎗䎑䎔䎔䎔䎓䎑䎗 䎜䎛䎑䎓 䎔䎓䎕䎑䎛䎔䎓䎘䎑䎘 䎜䎗䎑䎙 䎔䎓䎓䎑䎘䎔䎕䎖䎑䎔 䎔䎓䎖䎑䎔 䎔䎓䎜䎑䎓䎘䎙䎑䎜 䎘䎕䎑䎛 䎜䎜䎑䎘䎘䎚䎑䎕 䎙䎘䎑䎕 䎜䎛䎑䎓䎘䎘䎑䎙 䎕䎚䎑䎕 䎛䎚䎑䎚䎘䎚䎑䎛 䎙䎙䎑䎓 䎔䎔䎕䎑䎙䎔䎕䎑䎕 䎔䎘䎑䎙 䎔䎕䎑䎓䎔䎕䎑䎓 䎔䎙䎑䎚 䎔䎕䎑䎓䎔䎓䎑䎗 䎔䎖䎑䎔 䎔䎓䎑䎙䎔䎗䎑䎕 䎔䎚䎑䎓 䎔䎖䎑䎘䎔䎓䎑䎖䎚䎑䎕 䎔䎗䎑䎗
䎔䎔䎑䎓
䎚䎑䎔
䎜䎑䎕
䎛䎑䎚
䎘䎑䎜
䎜䎑䎕
䎔䎔䎑䎖
䎛䎑䎙 䎕䎗䎑䎚
䎛䎜䎑䎔 䎘䎜䎑䎔 䎚䎚䎑䎓䎜䎓䎑䎙 䎘䎜䎑䎙 䎚䎜䎑䎙䎚䎛䎑䎚 䎘䎓䎑䎗 䎙䎗䎑䎜䎜䎛䎑䎕 䎙䎚䎑䎘 䎛䎙䎑䎘
䎙䎑䎜
䎘䎑䎙
䎘䎑䎙
䎙䎑䎖
䎘䎑䎘
䎘䎑䎖
䎘䎑䎗
䎗䎑䎘
䎘䎑䎖
䎛䎑䎜
䎙䎑䎜
䎙䎑䎕
䎛䎘䎑䎔 䎚䎖䎑䎜 䎜䎓䎑䎜䎛䎚䎑䎕 䎚䎔䎑䎜 䎜䎕䎑䎗䎙䎜䎑䎔 䎙䎜䎑䎕 䎛䎘䎑䎜䎜䎛䎑䎜 䎛䎓䎑䎙 䎜䎗䎑䎗䎘䎜䎑䎘 䎙䎙䎑䎖 䎖䎜䎑䎛䎙䎜䎑䎗 䎚䎔䎑䎛 䎗䎗䎑䎚䎕䎙䎑䎔 䎗䎓䎑䎔 䎖䎘䎑䎛䎛䎖䎑䎔 䎛䎚䎑䎓 䎗䎗䎑䎚
䎛䎑䎗
䎛䎑䎓
䎛䎑䎔
䎛䎑䎕
䎛䎑䎔
䎛䎑䎙
䎙䎑䎕
䎚䎑䎛
䎙䎑䎚
䎔䎓䎑䎚
䎛䎑䎕
䎛䎑䎛

38
58

ENV/JM/MONO(2011)39

Fig. 4. Distribution of the viability for each chemical.

39
59

ENV/JM/MONO(2011)39

Fig. 4. continued

40
60

ENV/JM/MONO(2011)39

㩷Fig. 4. continued.
Table 14. Viability of chemicals each laboratory by 3rd phase study䎦䏋䏈䏐䎑 䎹䏌䏙䏒 䎶䏆䏒䏕䏈 䎨䏛䏓䎑

䎤

䏑䏒

䎕

䎥

䎦䏄䏗䏈䏊
䎕䎑䎚
䏒䏕䏜䎃䎕

䎦

䎦䏄䏗䏈䏊
䏒䏕䏜䎃䎕

䎧

䎦䏄䏗䏈䏊
䎖䎑䎖
䏒䏕䏜䎃䎕

䎨

䎦䏄䏗䏈䏊
䎖䎑䎖
䏒䏕䏜䎃䎕

䎩

䎦䏄䏗䏈䏊
䏒䏕䏜䎃䎕

䎖

䎗

䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖

䏄
䎔䎖䎑䎖
䎔䎗䎑䎕
䎔䎗䎑䎓
䎔䎑䎘
䎖䎑䎔
䎔䎑䎘
䎓䎑䎚
䎔䎑䎖
䎓䎑䎘
䎔䎗䎑䎘
䎔䎖䎑䎙
䎔䎛䎑䎙
䎖䎑䎜
䎗䎑䎘
䎔䎑䎛
䎘䎑䎙
䎘䎑䎚
䎘䎑䎗

䏅
䎔䎔䎑䎛
䎔䎓䎑䎕
䎔䎔䎑䎔
䎕䎑䎕
䎕䎑䎕
䎕䎑䎘
䎓䎑䎚
䎔䎑䎔
䎓䎑䎛
䎕䎗䎑䎓
䎔䎙䎑䎓
䎔䎘䎑䎘
䎖䎑䎗
䎕䎑䎚
䎖䎑䎘
䎚䎑䎕
䎙䎑䎔
䎗䎑䎕

䎯䏄䏅䎑
䏆
䏇
䎔䎖䎑䎕 䎔䎖䎑䎛䎕䎕䎑䎘䎜䎑䎜䎔䎕䎑䎖 䎔䎖䎑䎕
䎕䎑䎘
䎗䎑䎓
䎕䎑䎜
䎖䎑䎓
䎖䎑䎓
䎖䎑䎜
䎓䎑䎚
䎙䎑䎜
䎔䎑䎗
䎕䎑䎓
䎔䎑䎓
䎓䎑䎛
䎔䎕䎑䎚 䎔䎓䎑䎖䎔䎕䎑䎘 䎔䎛䎑䎖䎔䎕䎑䎙 䎕䎖䎑䎓
䎖䎑䎗
䎛䎑䎕
䎖䎑䎖
䎖䎑䎜
䎖䎑䎘
䎖䎑䎚
䎙䎑䎘
䎙䎑䎗
䎙䎑䎛
䎘䎑䎗
䎙䎑䎘
䎘䎑䎗

䏉
䎔䎔䎑䎗
䎔䎔䎑䎖
䎔䎗䎑䎖
䎔䎑䎚
䎕䎑䎙
䎖䎑䎕
䎓䎑䎛
䎗䎑䎛
䎔䎑䎓
䎔䎖䎑䎛
䎛䎑䎛
䎔䎜䎑䎕
䎖䎑䎕
䎗䎑䎕
䎘䎑䎓
䎘䎑䎕
䎚䎑䎗
䎘䎑䎓

䏊
䎔䎖䎑䎚
䎛䎑䎚
䎔䎗䎑䎖
䎖䎑䎜
䎖䎑䎚
䎗䎑䎚
䎔䎑䎓
䎓䎑䎗
䎓䎑䎖
䎔䎜䎑䎖
䎔䎘䎑䎕
䎔䎗䎑䎔
䎗䎑䎔
䎖䎑䎔
䎘䎑䎔
䎚䎑䎕
䎙䎑䎛
䎚䎑䎙

41
61

ENV/JM/MONO(2011)39
Table 15㩷 Summary statistical analysis of viability each chemical by 3rd phase study䎯䏄䏅䎑䎦䏋䏈䏐䎑 䎶䏗䏄䏗䎑
䏄
䏅
䏆
䏇
䏉
䏊
䎰䏈䏄䏑
䎔䎖䎑䎛
䎔䎔䎑䎓
䎔䎙䎑䎓
䎔䎕䎑䎖
䎔䎕䎑䎖
䎔䎕䎑䎕
䎰䏈䏇䏌䏄䏑
䎔䎗䎑䎓
䎔䎔䎑䎔
䎔䎖䎑䎕
䎔䎖䎑䎕
䎔䎔䎑䎗
䎔䎖䎑䎚
䎤
䎰䏌䏑
䎔䎖䎑䎖
䎔䎓䎑䎕
䎔䎕䎑䎖
䎜䎑䎜
䎔䎔䎑䎖
䎛䎑䎚
䎰䏄䏛
䎔䎗䎑䎕
䎔䎔䎑䎛
䎕䎕䎑䎘
䎔䎖䎑䎛
䎔䎗䎑䎖
䎔䎗䎑䎖
䎰䏈䏄䏑
䎕䎑䎓
䎕䎑䎖
䎕䎑䎛
䎖䎑䎙
䎕䎑䎘
䎗䎑䎔
䎰䏈䏇䏌䏄䏑
䎔䎑䎘
䎕䎑䎕
䎕䎑䎜
䎖䎑䎜
䎕䎑䎙
䎖䎑䎜
䎥
䎰䏌䏑
䎔䎑䎘
䎕䎑䎕
䎕䎑䎘
䎖䎑䎓
䎔䎑䎚
䎖䎑䎚
䎰䏄䏛
䎖䎑䎔
䎕䎑䎘
䎖䎑䎓
䎗䎑䎓
䎖䎑䎕
䎗䎑䎚
䎰䏈䏄䏑
䎓䎑䎛
䎓䎑䎛
䎔䎑䎓
䎖䎑䎕
䎕䎑䎕
䎓䎑䎙
䎰䏈䏇䏌䏄䏑
䎓䎑䎚
䎓䎑䎛
䎔䎑䎓
䎕䎑䎓
䎔䎑䎓
䎓䎑䎗
䎦
䎰䏌䏑
䎓䎑䎘
䎓䎑䎚
䎓䎑䎚
䎓䎑䎛
䎓䎑䎛
䎓䎑䎖
䎰䏄䏛
䎔䎑䎖
䎔䎑䎔
䎔䎑䎗
䎙䎑䎜
䎗䎑䎛
䎔䎑䎓
䎰䏈䏄䏑
䎔䎘䎑䎙
䎔䎛䎑䎘
䎔䎕䎑䎙
䎔䎚䎑䎕
䎔䎖䎑䎜
䎔䎙䎑䎕
䎰䏈䏇䏌䏄䏑
䎔䎗䎑䎘
䎔䎙䎑䎓
䎔䎕䎑䎙
䎔䎛䎑䎖
䎔䎖䎑䎛
䎔䎘䎑䎕
䎧
䎰䏌䏑
䎔䎖䎑䎙
䎔䎘䎑䎘
䎔䎕䎑䎘
䎔䎓䎑䎖
䎛䎑䎛
䎔䎗䎑䎔
䎰䏄䏛
䎔䎛䎑䎙
䎕䎗䎑䎓
䎔䎕䎑䎚
䎕䎖䎑䎓
䎔䎜䎑䎕
䎔䎜䎑䎖
䎰䏈䏄䏑
䎖䎑䎗
䎖䎑䎕
䎖䎑䎗
䎘䎑䎖
䎗䎑䎕
䎗䎑䎔
䎰䏈䏇䏌䏄䏑
䎖䎑䎜
䎖䎑䎗
䎖䎑䎗
䎖䎑䎜
䎗䎑䎕
䎗䎑䎔
䎨
䎰䏌䏑
䎔䎑䎛
䎕䎑䎚
䎖䎑䎖
䎖䎑䎚
䎖䎑䎕
䎖䎑䎗
䎰䏄䏛
䎗䎑䎘
䎖䎑䎘
䎖䎑䎘
䎛䎑䎕
䎘䎑䎓
䎘䎑䎔
䎰䏈䏄䏑
䎘䎑䎘
䎘䎑䎛
䎙䎑䎙
䎘䎑䎚
䎘䎑䎜
䎚䎑䎕
䎰䏈䏇䏌䏄䏑
䎘䎑䎙
䎙䎑䎔
䎙䎑䎘
䎘䎑䎗
䎘䎑䎕
䎚䎑䎕
䎩
䎰䏌䏑
䎘䎑䎗
䎗䎑䎕
䎙䎑䎘
䎘䎑䎗
䎘䎑䎓
䎙䎑䎛
䎰䏄䏛
䎘䎑䎚
䎚䎑䎕
䎙䎑䎛
䎙䎑䎗
䎚䎑䎗
䎚䎑䎙

42
62

ENV/JM/MONO(2011)39

Fig.5 Distribution of viability each chemical
8-2-5. IL-1Į
36.
The results of the LabCyte EPI-MODEL 24 skin irritation test when IL-1Į was evaluated as anindicator are summarized in Table 16.
Table 16. IL-1Į levels from each laboratory.
䎦䏋䏈䏐䎑 䎪䎫䎶 䎶䏆䏒䏕䏈 䎨䏛䏓䎑

䎓䎔

䏑䏒

䎓

䎓䎕

䏑䏒

䎓

䎓䎖

䏑䏒

䎓

䎓䎗

䏑䏒

䎓

䎓䎘

䏑䏒

䎓䎑䎖

䎓䎙

䏑䏒

䎓䎑䎖

䎓䎚

䏑䏒

䎔

䎓䎛

䏑䏒

䎔

䎓䎜

䏑䏒

䎔䎑䎚

䎔䎓

䏑䏒

䎔䎑䎚

䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖

䏄
䎑
䎑
䎑

䏅
䎑
䎑
䎑

䎯䏄䏅䎑
䏇

䏆
䎑
䎑
䎑

䎑
䎑
䎑

䎔䎖䎕䎑䎛
䎙䎛䎑䎔
䎜䎚䎑䎙
䎔䎕
䎚䎑䎔
䎔䎓䎑䎚
䎔䎓
䎘䎑䎖
䎙䎑䎖
䎔䎕䎕
䎖䎘䎑䎚
䎗䎗䎑䎗
䎘䎜
䎙䎕䎑䎜
䎙䎛䎑䎛

䏈
䎑
䎑
䎑

䎘䎕䎑䎜 䎘䎜䎑䎖 䎗䎔䎑䎕䎘䎙䎑䎘䎖䎚 䎛䎜䎑䎔䎗䎔䎑䎔䎚䎙 䎚䎕䎑䎗䎜䎑䎘 䎔䎘䎑䎘
䎛䎑䎙䎛䎑䎙 䎔䎔䎑䎚 䎔䎜䎑䎜䎔䎓䎑䎖 䎔䎕䎑䎜䎜䎑䎗䎙䎛 䎔䎔䎑䎚
䎛䎘䎑䎘 䎔䎖䎑䎕
䎗䎑䎚
䎚䎑䎕
䎚䎑䎜
䎜䎚䎑䎙 䎕䎗䎑䎖 䎛䎔䎑䎕䎙䎖䎑䎘 䎖䎘䎑䎔 䎔䎔䎘䎑䎖䎕䎙 䎖䎔䎑䎕 䎗䎜䎑䎗䎛䎘䎑䎚䎔䎔䎗 䎛䎘䎑䎙
䎜䎖䎑䎙 䎔䎓䎗䎑䎜 䎔䎖䎜䎑䎘䎛䎘䎑䎔 䎛䎕䎑䎜 䎙䎗䎑䎘
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎛䎑䎕
䎜䎑䎗 䎛䎗䎑䎔䎗䎑䎔䎖䎑䎙䎙䎑䎗 䎖䎔䎑䎙 䎔䎓䎑䎗䎙䎗䎑䎔 䎖䎖䎑䎔
䎘䎑䎕䎔䎓䎑䎜 䎔䎚䎑䎔 䎔䎔䎑䎕 䎗䎕䎑䎙䎔䎜䎑䎛䎛䎑䎛䎛䎑䎛 䎖䎕䎑䎕䎖䎔䎑䎖䎙䎑䎛 䎕䎓䎑䎔 䎕䎔䎑䎖䎕䎚䎑䎜䎚䎑䎗 䎖䎔䎑䎖 䎗䎔䎑䎕䎔䎚䎑䎔 䎔䎕䎑䎚䎔䎘 䎘䎓䎑䎗
䎙䎙䎑䎕 䎔䎕䎑䎕䎖䎓 䎗䎕䎑䎔

䏉
䎑
䎑
䎑

䏊
䎑
䎑
䎑

䎙䎓䎑䎚 䎙䎔䎑䎖䎜䎑䎗䎙䎛䎑䎗 䎜䎜䎑䎖䎜䎑䎙䎗䎙 䎚䎓䎑䎔 䎔䎕䎑䎙䎕䎖䎑䎕 䎔䎕䎑䎚䎛䎑䎔䎔䎓䎑䎘䎜䎑䎕 䎔䎔䎑䎜䎔䎔䎑䎖䎙䎑䎚 䎔䎘䎑䎚
䎜䎑䎘
䎕䎑䎘
䎙䎑䎖
䎔䎘䎑䎔
䎕䎑䎙
䎛䎑䎙
䎜䎑䎚
䎖䎑䎗
䎙䎑䎛
䎘䎚䎑䎚 䎔䎛䎖䎑䎘 䎔䎘䎑䎗䎖䎙䎑䎙 䎑䎕䎛䎑䎘䎖䎖 䎔䎜䎔䎑䎙 䎖䎖䎑䎕䎜䎗䎑䎗 䎙䎓䎑䎛 䎔䎔䎕䎑䎘䎛䎔䎑䎗 䎗䎛䎑䎔 䎙䎕䎑䎔䎘䎕䎑䎜 䎘䎗䎑䎛 䎔䎗䎚䎑䎔
䎑
䎑
䎑
䎑
䎑
䎑
䎙䎑䎜 䎕䎔䎑䎗
䎘䎑䎖
䎛䎑䎘
䎗䎑䎜
䎘䎑䎛
䎙䎑䎚
䎕䎑䎔
䎚䎑䎕
䎕䎜䎑䎘
䎖䎖
䎚䎑䎗
䎙䎑䎘 䎕䎘䎑䎖
䎜䎑䎚䎔䎔䎑䎕 䎕䎗䎑䎚 䎔䎓䎑䎙䎗䎙䎑䎘 䎖䎜䎑䎖䎜䎑䎛䎕䎙䎑䎚 䎕䎙䎑䎚 䎔䎗䎑䎘䎕䎙䎑䎖 䎕䎗䎑䎕 䎔䎖䎑䎕

43
63

ENV/JM/MONO(2011)39
Table 16. continued.
䎦䏋䏈䏐䎑

䎪䎫䎶

䎶䏆䏒䏕䏈 䎨䏛䏓䎑

䎔䎔

䏑䏒

䎕

䎔䎕

䏑䏒

䎕

䎔䎗

䎦䏄䏗䏈䏊䏒䏕䏜
䎕

䎕䎑䎖

䎔䎘

䎦䏄䏗䏈䏊䏒䏕䏜
䎕

䎕䎑䎖

䎔䎙

䎦䏄䏗䏈䏊䏒䏕䏜
䎕

䎕䎑䎚

䎔䎚

䎦䏄䏗䏈䏊䏒䏕䏜
䎕

䎕䎑䎚

䎔䎛

䎦䏄䏗䏈䏊䏒䏕䏜䎕䎖䎔䎜䎦䏄䏗䏈䏊䏒䏕䏜䎕䎖䎕䎓䎦䏄䏗䏈䏊䏒䏕䏜
䎕

䎗

䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖

䎯䏄䏅䎑
䏇
䏈
䏉
䎘
䎔䎛 䎔䎘䎑䎖 䎔䎓䎑䎗 䎔䎙䎑䎕
䎖䎑䎖
䎔䎘䎑䎛
䎔䎜
䎜䎑䎚
䎛䎑䎔
䎔䎛䎑䎕
䎛䎑䎜
䎛䎑䎚
䎛䎑䎙 䎔䎕䎑䎙䎑䎑䎑䎔䎘䎚䎑䎕 䎔䎕䎓䎑䎗 䎑䎑䎑䎑䎔䎔䎖 䎔䎔䎛䎑䎙 䎜䎓䎑䎕䎑䎑䎑䎑䎘䎛䎑䎖 䎙䎙䎑䎕
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎛䎙䎑䎜 䎙䎛䎑䎔 䎔䎕䎜䎑䎗 䎑䎔䎕䎙䎑䎛 䎔䎔䎙䎑䎘䎑䎔䎓䎓䎑䎕 䎚䎗䎑䎗 䎔䎙䎜䎑䎚 䎚䎙䎑䎔 䎔䎓䎚䎑䎘䎔䎕䎔䎑䎕 䎗䎕䎑䎘 䎛䎖䎑䎙 䎑䎚䎖䎑䎔 䎛䎚䎑䎖
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎙䎔䎑䎘 䎑
䎙䎓䎑䎙 䎜䎓䎑䎖 䎛䎙䎑䎜 䎔䎔䎗䎑䎘䎘䎚䎑䎚 䎔䎓䎗䎑䎜 䎗䎘䎑䎛 䎕䎕䎔䎑䎖 䎜䎛䎑䎚 䎚䎙䎑䎗䎑䎔䎚䎑䎕 䎘䎔䎑䎗 䎔䎖䎛䎑䎔 䎙䎖䎑䎜 䎔䎓䎕䎑䎕䎑䎘䎚䎑䎖 䎑
䎑䎔䎓䎜䎑䎕 䎑䎑䎑䎑䎑䎑䎑䎙䎜䎑䎕 䎑
䎔䎓䎕䎑䎖 䎑䎑䎑䎙䎛 䎘䎜䎑䎘 䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䏄

䏅
䎖䎔䎑䎔
䎔䎔䎑䎜
䎘

䏆

䏊
䎙䎑䎗
䎚䎑䎘
䎔䎔䎑䎜
䎖䎗䎑䎘
䎕䎚䎑䎖
䎔䎖䎑䎙

䎜䎓䎑䎛
䎚䎓䎑䎜
䎚䎜䎑䎕

䎔䎛
䎗䎘䎑䎔
䎕䎕䎑䎔

Cells highlighted in yellow indicate that the classification changed based on the IL-1Į data.

4464

ENV/JM/MONO(2011)39
8-2-6. Classification of three independent viabilities at each laboratory
37.
The classifications from mean of three independent viabilities only evaluated MTT assay wereshown in Table 17 in 2nd phase study and Table 19 in 3rd phase study. Refer to Table 18, the IL-1Į resultschanged the classification for only 3 data points. The classification of Allyl phenoxy-acetate by Lab f waschanged the misunderstood classification. The other two chemicals were changed the correct classification.
Regarding the ILĮ only a few chemicals showed different results but the overall call was that ILĮ did not
significantly contribute to the performance of the assay.

Table 17.Classification using three independent viabilities by 2nd phase study
䇸P䇹:Positive䇮䇸N䇹: Negative
㩷
Chem.
01
02
03
04
05
06
07
08
09
10
11
12
14
15
16
17
18
19
20

GHS
no
no
no
no
no
no
no
no
no
no
no
no
Category 2
Category 2
Category 2
Category 2
Category 2
Category 2
Category 2

Score
0
0
0
0
0.3
0.3
1
1
1.7
1.7
2
2
2.3
2.3
2.7
2.7
3
3
4

Lab.
a
P
N
N
N
N
N
P
N
N
N
N
P
P
P
N
P
N
P
P

b
P
N
N
N
N
N
P
N
N
N
N
P
P
P
N
P
N
P
P

c
P
N
N
N
N
N
P
N
N
N
N
P
P
P
N
P
N
P
P

d
P
N
N
N
N
N
P
N
N
N
N
N
P
P
P
P
N
P
P

e
P
N
N
N
N
N
P
N
N
N
N
N
P
P
N
P
N
N
P

f
P
N
N
N
N
N
P
N
N
N
N
N
P
P
N
P
N
N
P

g
P
N
N
N
N
N
P
N
N
N
N
N
P
P
N
P
N
P
P

Table.18. Classification of chemicals by MTT assay demolished by additional IL-1Į measurement
Lab. Classific Classific
In vivoCAS
ation by ation by
No. Chemical
GHS label score
MTT+IL
number
MTT
(PII)
-1Į
assay
N
P
f
05 Allyl phenoxy-acetate
7493-74-5 no0.3
16 1-bromohexane

111-25-1

Category 2

2.7

a

N

P

18 di-n-propyl disulphide629-19-6

Category 2

3

d

N

P

45
65

ENV/JM/MONO(2011)39
Table 19 Classification using three independent viabilities by 3rd phase study
䇸P䇹:Positive䇮䇸N䇹: Negative
㩷
Lab.
Chem.
in vivo
Score
a
b
c
d
f
P
P
P
P
P
A
no
2
B

Category 2

2.7

C

Category 2

2.7

DCategory 2

3.3

E

Category 2

3.3

F

Category 2

4

g
P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

Table 20.㩷 Sensitivity, specificity and accuracy on MTT assay vs GHS-EU classification in the 2nd + 3rd
Phase validation study (25 substances)䎬䏑䏇䏈䏛䎶䏈䏑䏖䏌䏗䏌䏙䏌䏗䏜䎶䏓䏈䏆䏌䏉䏌䏆䏌䏗䏜䎤䏆䏆䏘䏕䏄䏆䏜

䎯䏄䏅䎑
䏄
䏅
䏆
䏇
䏉
䏊
䎔䎓䎒䎔䎕 䎔䎓䎒䎔䎕 䎔䎓䎒䎔䎕 䎔䎔䎒䎔䎕 䎜䎒䎔䎕 䎔䎓䎒䎔䎕䎛䎖䎑䎖 䎛䎖䎑䎖 䎛䎖䎑䎖 䎜䎔䎑䎙䎚䎘䎛䎖䎑䎖䎜䎒䎔䎖 䎜䎒䎔䎖 䎜䎒䎔䎖 䎔䎓䎒䎔䎖 䎔䎓䎒䎔䎖 䎔䎓䎒䎔䎖䎙䎜䎑䎕 䎙䎜䎑䎕 䎙䎜䎑䎕 䎚䎙䎑䎜 䎚䎙䎑䎜 䎚䎙䎑䎜䎔䎜䎒䎕䎘 䎔䎜䎒䎕䎘 䎔䎜䎒䎕䎘 䎕䎔䎒䎕䎘 䎔䎜䎒䎕䎘 䎕䎓䎒䎕䎘
䎚䎙
䎚䎙
䎚䎙
䎛䎗
䎚䎙
䎛䎓

Table 21.㩷 Sensitivity, specificity and accuracy on MTT assay vs GHS-EU classification in 2nd phase study(19 substances).

䎬䏑䏇䏈䏛䎶䏈䏑䏖䏌䏗䏌䏙䏌䏗䏜䎶䏓䏈䏆䏌䏉䏌䏆䏌䏗䏜䎤䏆䏆䏘䏕䏄䏆䏜

䎯䏄䏅䎑
䏄
䏅
䏆
䏇
䏈
䏉
䏊
䎘䎒䎚
䎘䎒䎚
䎘䎒䎚
䎙䎒䎚
䎗䎒䎚
䎗䎒䎚
䎘䎒䎚
䎚䎔䎑䎗 䎚䎔䎑䎗 䎚䎔䎑䎗 䎛䎘䎑䎚 䎘䎚䎑䎔 䎘䎚䎑䎔 䎚䎔䎑䎗䎜䎒䎔䎕 䎜䎒䎔䎕 䎜䎒䎔䎕 䎔䎓䎒䎔䎕 䎔䎓䎒䎔䎕 䎔䎓䎒䎔䎕 䎔䎓䎒䎔䎕䎚䎘䎚䎘䎚䎘䎛䎖䎑䎖 䎛䎖䎑䎖 䎛䎖䎑䎖 䎛䎖䎑䎖䎔䎗䎒䎔䎜 䎔䎗䎒䎔䎜 䎔䎗䎒䎔䎜 䎔䎙䎒䎔䎜 䎔䎗䎒䎔䎜 䎔䎗䎒䎔䎜 䎔䎘䎒䎔䎜䎚䎖䎑䎚 䎚䎖䎑䎚 䎚䎖䎑䎚 䎛䎗䎑䎕 䎚䎖䎑䎚 䎚䎖䎑䎚 䎚䎛䎑䎜

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Table 22.㩷 Sensitivity, specificity and accuracy of the MTT assay and IL-1Į vs. the GHS-EU classificationin 2nd phase study (19 substances).

䎬䏑䏇䏈䏛䎶䏈䏑䏖䏌䏗䏌䏙䏌䏗䏜䎶䏓䏈䏆䏌䏉䏌䏆䏌䏗䏜䎤䏆䏆䏘䏕䏄䏆䏜

䎯䏄䏅䎑
䏄
䏅
䏆
䏇
䏈
䏉
䏊
䎙䎒䎚
䎘䎒䎚
䎘䎒䎚
䎚䎒䎚
䎗䎒䎚
䎗䎒䎚
䎘䎒䎚
䎛䎘䎑䎚 䎚䎔䎑䎗 䎚䎔䎑䎗䎔䎓䎓䎘䎚䎑䎔 䎘䎚䎑䎔 䎚䎔䎑䎗䎜䎒䎔䎕 䎜䎒䎔䎕 䎜䎒䎔䎕 䎔䎓䎒䎔䎕 䎔䎓䎒䎔䎕 䎜䎒䎔䎕 䎔䎓䎒䎔䎕䎚䎘䎚䎘䎚䎘䎛䎖䎑䎖 䎛䎖䎑䎖䎚䎘䎛䎖䎑䎖䎔䎘䎒䎔䎜 䎔䎗䎒䎔䎜 䎔䎗䎒䎔䎜 䎔䎚䎒䎔䎜 䎔䎗䎒䎔䎜 䎔䎖䎒䎔䎜 䎔䎘䎒䎔䎜䎚䎛䎑䎜 䎚䎖䎑䎚 䎚䎖䎑䎚 䎛䎜䎑䎘 䎚䎖䎑䎚 䎙䎛䎑䎗 䎚䎛䎑䎜

Table 23(A). Mean and range of Sensitivity, specificity and accuracy on the MTT assay using LabCyte
EPI-MODEL vs. GHS-EU classification in the 2nd + 3rd Phase validation study (25substances)
N
Mean
Min.
Max.
ECVAM criteria
Sensivitity (%)
6
83.3
75.0
91.6
80.0
Specificity (%)
6
73.1
69.2
76.9
70.0
Accuracy (%)
6
78.0
76.0
84.0
75.0

Table 23(B). Mean and range of sensitivity, specificity and accuracy of the MTT assay vs. the GHS-EU
classification in 2nd phase study (19 substances).
N
Mean
Min.
Max.
ECVAM criteria
Sensivitity (%)
7
69.4
57.1
85.7
80.0
Specificity (%)
7
79.7
75.0
83.3
70.0
Accuracy (%)
7
75.9
73.7
84.2
75.0
Table 23(C). Mean and range of sensitivity, specificity and accuracy of the MTT assay and IL-1Į vs. the
GHS-EU classification in 2nd phase study (19 substances).
N
Mean
Min.
Max.
ECVAM criteria
Sensivitity (%)
7
73.4
57.1
100.0
80.0
Specificity (%)
7
78.6
69.2
76.9
70.0
Accuracy (%)
7
76.7
68.4
89.5
75.0
9. Discussion
9-1. Reliability38.
All data of negative control and positive control each laboratory in 2nd and 3rd phase study was
sufficient with the acceptance criteria as shown in Tables 10 and 11. There were high respectabilities within
and between laboratories in this model.
39.
In all data, Invalid data obtained only one data (Lab a, run 1). This lab performed at retesting andwe accepted data of run 2-4. Therefore, the rate of invalid at this assay is 0.2% (total 1/508, 400 data: 3runs
X 7 labs X 19 chemicals+1 run in 2nd phase study & 108 data; 3 runs X 6 labs X 6 chemicals in 3rd phasestudy ). Based on a comparison of the results from the seven laboratories, the classification of 3 chemicals(No. 12, 16 and 19) should be potentially changed. However, the classifications of the remaining chemicals
were not changed. The variations of these chemicals and No.18 are larger than those of others. The IL-1Į
data changed the classification for No. 5, 16 and 18 at Lab. f (No. 5), Lab. a (No. 16), and Lab. d (No. 18).
The effect of IL-1Į on the reliability of these results is small.
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9-2. Predictivity
40.
In December 2008, the EU adopted the UN Globally Harmonised System for Classification andLabelling and will implement this by means of the so-called CLP regulation (Regulation EC 1272/2008).
The new EU classification system based on UN GHS (abbreviated here as "GHS-EU") continues to use twocategories to distinguish non-irritant (no-category) from irritant (category 2) substances. However, according
to the new rules for skin irritation classification and labelling, the cut-off score to distinguish between nocategory and category 2 substances was shifted to 2.3 from a value of 2.0 (EU classification system).
Consequently substances with an in vivo score between 2.0 and 2.3 that are considered irritant under the
existing EU classification system will be considered non-irritants under the future GHS-EU classification
system, which does not use the optional UN GHS category 3.
41.
The prediction values of the LabCyte EPI-MODEL 24 skin irritation test when it was evaluated bycell viabilities (MTT) as an indicator, and the GHS-EU classifications are shown in Table 20. The sensitivity,
specificity and accuracy of this prediction model at each laboratory were 75-91.6 %, 69.2-76.9 %, and 7684 %, respectively. These predictivities were similar with each laboratory. The mean and range of prediction
values of the skin irritation test with LabCyte EPI-MODEL 24 when it was only evaluated by MTT as anindicator and the GHS-EU classification are shown in Table 23(A). The mean sensitivity, specificity andaccuracy of this prediction model are 83.3%, 73.1%, and 78.0%, respectively. Some deviations from the
ESAC Performance standard (sensitivity of 80%, specificity of 70% and an accuracy of 75%) that were
specific adaptations for the Japanese model.The effect of IL-1Į on the predictivity was small compared withresults in Tables 21,22, 23 (B) and 23(c).
G
10. Conclusions
42.
Based on the GHS-EU classification, 12 irritants and 13 non-irritants in the ECVAM PerformanceStandards(2007,2009) were tested by the 7 labs using LabCyte EPI-MODEL. The assay demonstrated highreliability within and between laboratories, and acceptable reliability of the positive control (100%) andaccuracy (77.5% overall accuracy, 82.3% overall sensitivity, 72.6% overall specificity) on the MTT assay for
use as a stand-alone assay to distinguish between skin irritants and non-irritants.
This report summarized at JSAAE 1st report and 2nd report on this validation study.
11. Acknowledgement
43.

This validation study has supported by the Health and Labour Sciences Research Grant, Japan.

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